Paeonol prevents IL-1β-induced inflammatory response and degradation of type II collagen in human primary chondrocytes

Abstract Osteoarthritis (OA) is a common joint disease for which a safe and reliable treatment has yet to be developed. Here, we demonstrated the potential benefit of treatment with paeonol, a derivative of Paeonia suffruticosa, in the treatment and prevention of OA. Chondrogenic cell line ATDC5 cells were cultured with IL-1β and the effects of paeonol were assessed through qRT-PCR, western blot analysis, MTT, ELISA, and NF-κB luciferase reporter gene assay. Our findings demonstrate a novel ability of paeonol to inhibit numerous factors of OA, including expressions of IL-6, TNF-α, NOX2, PTGS2, NUCB2/nesfatin-1, ICAM-1, VCAM-1, MMP-3/13, degradation of type II collagen, and NF-κB activation through the rescue of IκBα. Additionally, we assessed the effects of paeonol on cell viability to confirm its safety. These findings implicate a valuable potential role of paeonol in the treatment and prevention of OA.


Introduction
Although osteoarthritis (OA) is a common debilitating disease worldwide, the pathophysiology of OA remains poorly understood. Overexpression of cytokines such as IL-1b plays a key role in the pathogenesis of OA by upregulating expression of degradative enzymes and proinflammatory cytokines, among other things [1]. One of the major hallmarks of OA is excessive loss of extracellular matrix (ECM) in the articular tissues, including type II collagen, which provides cartilage tissue with structural rigidity, thereby allowing joints to withstand heavy mechanical loading. It is degraded by matrix metalloproteinases (MMPs), which are upregulated in OA chondrocytes [2,3]. MMP-3 and MMP-13 are shown to induce degradation of type II collagen [4]. Additionally, MMP-3 induces expression of various pro-MMPs, including pro-MMP-13 [5]. MMP-13 is reported to specifically cleave type II collagen, making it a valuable target for therapies against cartilage degradation [6].
Another major factor driving the development and progression of OA is oxidative stress. The NADPH oxidase isoform NOX2 has been shown to induce the generation of ROS upon incubation with IL-1b. While the role of NOX2 in OA has not yet been fully elucidated, a recent study using human chondrogenic cell line ATDC5 cells showed that NOX2 may be involved in the loss of articular ECM by the activation of hyaluronidase through intra-and extra-cellular acidification [7]. Prostaglandin-endoperoxide synthase 2 (PTGS2) is an isoenzyme produced during inflammation that has been shown to regulate prostaglandin biosynthesis and pain response in OA [8]. Inhibition of PTGS2 has been considered as a potential method for managing pain and inflammation in OA [9]. The adipokine nucleobindin 2 (NUCB2/ nesfatin-1) is a calcium-binding protein that regulates calcium homoeostasis and inflammation by inducing IL-6 and IL-8 when administered in combination with IL-1 in ATDC5 chondrocytes [10]. As a recent discovery in chondrocytes, there exists very little information on the function of NUCB2/nesfatin-1 in OA.
Numerous investigations have revealed the participation of proinflammatory cytokines in the pathological development of OA by driving expression of MMPs and degradative enzymes [11][12][13][14]. Two vascular adhesion molecules, ICAM1 and VCAM1, are important for promoting the inflammation in OA synovial tissue by regulating the recruitment of leukocytes through the vascular wall and into inflamed synovial tissue, where they interact with other cell types [15]. Increased expression of ICAM1 and VCAM1 have been shown to be correlated with the development of knee OA [16,17]. Additionally, ICAM1 and VCAM1 have been associated with the senescence-associated secretory phenotype (SASP), which causes chondrocytes to lose their normal functionality and instead promote inflammation and expression of cytokines, chemokines, and MMPs, thus perpetuating the inflammatory response and driving degradation of ECM [18,19]. One of the most important factors in OA is the NF-jB signalling pathway. Activation of NF-jB governs the expression of numerous genes in joint destruction, inflammation and homoeostasis. NF-jB activation is triggered by cytokines and chemokines, activation of other signalling pathways, and byproducts of cartilage degradation. [20,21]. Phosphorylation of IjBa induced by IL-1b triggers translocation of the dimers of NF-jB, including p65 protein, to the nucleus where activation of NF-jB transcription occurs [22,23].
Paeonol is the active compound found in Paeonia lactiflora Pallas, Cynanchum paniculatum, and Paeonia suffruticosa Andr and was recently shown to have anti-inflammatory effects in a rheumatoid arthritis model using human fibroblast-like synoviocytes stimulated with IL-1b [24]. Here, we examined the roles of treatment with paeonol on IL-1b-induced expression of cytokines, chemokines, adhesion molecules, ECM degradation and activation of IjBa/NF-jB in chondrogenic cell line ATDC5 cells. Our findings demonstrate that paeonol has a significant protective role against these factors of OA, thereby suggesting the potential value of paeonol in OA treatment.

Cell cultures and treatments
ATDC5 cells were cultured in DMEM with 10% FBS and 1% P/S and allowed to differentiate into mature chondrocytes [25]. Paeonol was obtained from the National Institute for Food and Drug Control (Beijing, China). Cells were stimulated with IL-1b (10 ng/mL) with or without paeonol (50, 100 lM) for 24 h.

Quantitative real time PCR
RNA from ATDC5 cells was extracted using Qiazol (Qiagen, USA). The isolated RNA (2 lg) was reverse-transcribed into cDNA. Expression of target genes was determined using realtime PCR with an SYBR Green PCR Master Mix. The 2 ÀDDCt approach was applied to calculate the level of detected genes, using GAPDH as an internal control [26].

Measurement of cell viability
Viability of ATDC5 cells was measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). Briefly, MTT (5 mg/mL) was put into each well of cell culture. After incubation at 37 C for 4 h, the product formazan was dissolved in DMSO and OD values recorded at 570 nm were used to index the viability percentage [28].

NF-jB luciferase reporter gene assay
The activity of NF-jB was examined using an NF-jB luciferase reporter gene assay. Briefly, NF-jB promoter-luciferase and b-galactosidase plasmids (Clontech, USA) were transfected into ATDC5 cells [29]. 24 h later, stimulated with IL-1b (10 ng/mL) with or without paeonol (50, 100 lM) for 24 h. Cells were then lysed and centrifuged at 15,000Â g. Supernatants were then harvested and used to determine luciferase and b-galactosidase activity (Thermo Fisher Scientific, USA) on a luminometer. Luciferase activity of NF-jB in ATDC5 cells was normalized to b-galactosidase activity.

Statistical analysis
The results are demonstrated as means ± SEM. Comparisons between different groups were made using analysis of variance (ANOVA) method. P values < .05 was considered of statistical significance.

Determination of cell viability
The molecular structure of paeonol is shown in Figure 1. To measure the effects of paeonol on cell viability, human chondrocytic cell line ATDC5 cells were stimulated with 0, 0.1, 1, 10, 50, 100 and 500 lM paeonol for 48 h. As shown in Figure 2, the dose of 100 mM has a slight effect on cell viability, but only the dose of 500 mM paeonol elicited a significant decline in cell viability, thereby indicating that lower concentrations of paeonol are safe. Therefore, we chose the highest concentrations considered to be safe, 50 and 100 mM, for the following experiments.

Paeonol inhibits expression of proinflammatory cytokines
Proinflammatory cytokines play a vital role in the pathogenesis of OA. Thus, we investigated the effects of paeonol on IL-1b-induced IL-6 and TNF-a in ATDC5 chondrocytes. Briefly, cells were exposed to insult from IL-1b for 48 h with or without 50 and 100 mM paeonol. As shown in Figure 3(A), exposure to IL-1b increased TNF-a and IL-6 at the gene level by about 4.5-and 5.7-fold, respectively. However, paeonol reduced expression of these cytokines, with 100 mM paeonol decreasing TNF-a and IL-6 to only 1.6 and 2.1-fold, respectively. We further confirmed these effects of paeonol using ELISA analysis. As shown in Figure 3(B), treatment with paeonol significantly reduced IL-1b-induced TNF-a and IL-6, reducing the expression of both cytokines to roughly 2-fold basal levels. These findings implicate a strong anti-inflammatory property in chondrocytes treated with paeonol, suggesting that paeonol may be of use in the treatment of OA.

Paeonol attenuates expression of ICAM-1 and VCAM-1
Expressions of the adhesion molecules ICAM1 and VCAM1 have been shown to be elevated in OA and high levels of these molecules are associated with greater risk of knee or hip replacement [17,30]. To assess the effect of treatment with paeonol on IL-1b-induced expression of cellular adhesion molecules, ATDC5 chondrocytes were exposed to insult from IL-1b with or without 50 and 100 mM paeonol for 24 h. As shown in Figure 4, IL-1b induced an about 3-fold increase in expression of both ICAM1 and VCAM1, which was attenuated by paeonol. The dose of 100 mM significantly lowered the mRNA levels of the two cytokines to less than 2-fold baseline. Concordantly, western blot analysis revealed that IL-1b led to a more than 3-fold increase in protein expression, which was lowered to roughly 1.5-fold baseline by the higher dose of paeonol.

Paeonol reduces expression of MMP-3/13 and reduction of type II collagen
Expression of MMPs degrades type II collagen. To investigate the effect of paeonol on the expression of MMPs, ATDC5 cells were cultured with IL-1b (10 ng/mL) with or without paeonol (50, 100 mM) for 24 h. The results show that insult from IL-1b gave rise to approximately 10-and 12-fold increases in MMP-3 and MMP-13, respectively, at the gene level, which were reduced to 3-and 4-fold basal levels by paeonol ( Figure 5(A)). Similarly, as revealed by the results of ELISA in Figure 5(B), IL-1b increased MMP-3 and MMP-13 to 7-and 8-fold basal levels, which was reduced by paeonol to 2-and 3-fold basal levels  ( Figure 5(B)). Importantly, an insult from IL-1b obviously induced reduction of type II collagen, which was rescued to near basal levels by paeonol ( Figure 6). These findings suggest a new biological function of paeonol in preventing loss of type II collagen by suppressing MMP-3 and MMP-13.

Inhibits phosphorylation of IjBa and activation of NF-jB
Activation of the NF-jB pathway is strongly correlated with the inflammatory response in OA. To elucidate the effects of paeonol on this pathway, we assessed the phosphorylation of IjBa and activity of NF-jB. As demonstrated in Figure 7, an insult from IL-1b (10 ng/mL) increased phosphorylation of IjBa by more than 3.5-fold, which was decreased by paeonol to about 1.75-fold basal levels. Finally, we assessed the influence of paeonol on nuclear levels of p65 and subsequent luciferase activity of NF-jB. As shown in Figure 8, there was a remarkable increase in nuclear p65 and luciferase activity of NF-jB by IL-1b, which was reduced by paeonol.

Discussion
OA is a disease characterized by excessive degradation of cartilage, chronic inflammation, infiltration of synovial tissue by immune cells, and eventual irreversible joint destruction. In the pathophysiology of OA, proinflammatory cytokines and cellular signalling pathways contribute greatly to disease progression and to sustaining a mild chronic inflammatory state [31]. Therefore, inhibition of the release of proinflammatory cytokines and blockage of cellular signalling pathways is regarded as an attractive option for the management and treatment of OA. Paeonol is a commonly used compound in traditional Chinese medicine (TCM) and has been demonstrated to have antimicrobial, anticancer, antioxidant, analgesic, antipyretic and anti-inflammatory activities [24,32,33]. However, little is known regarding the potential of this natural therapeutic agent until recently. Here, we used human chondrogenic cell line ATDC5 cells cultured with IL-1b to explore the potential effects of paeonol treatment in OA. We confirmed the safety of treatment with paeonol by performing a cell viability assay, which showed that doses under  500 mM had little to no effect on the cell viability of ATDC5 cells (Figure 1). Additionally, our findings demonstrate that paeonol possesses anti-oxidative stress and anti-inflammatory effects by inhibiting expression of NOX2, PTGS2 and NUCB2/ nesfatin-1 in ATDC5 chondrocytes, which have been suggested as a potential treatment targets for OA [34][35][36]. This beneficial effect suggests a novel role of paeonol against the expression of chemokines and adipokines by OA chondrocytes.
Expression of cytokines, especially IL-1b, IL-6 and TNF-a, are often considered to be the main factors driving the pathogenesis of OA. Hence, blockade or inhibition of these is considered a valuable approach for the management of OA. In the present study, we used insult from IL-1b to simulate the condition of OA in ATDC5 chondrocytes as previously described [37]. Our findings demonstrate a remarkable ability of paeonol to inhibit TNF-a and IL-6 induced by IL-1b. Another factor driving the pathology of OA is the recruitment of monocytes to the synovium by ICAM-1 and VCAM-1, resulting in chronic low-grade inflammation [38]. Our findings show that paeonol significantly reduced ICAM-1 and VCAM-1 in ATDC5 cells. Thus, treatment with paeonol may be a safe therapeutic option for reducing the expression of these molecules in a myriad of diseases. To our knowledge, this study is the first to demonstrate the beneficial role of paeonol in inhibiting ICAM-1 and VCAM-1 in an OA chondrogenic cell model.
Perhaps the most prominent feature of OA, inhibition of impairment of the articular cartilage matrix is a primary treatment target. Here, we demonstrated the ability of paeonol to suppress MMP-3 and MMP-13 and further confirmed the ability of paeonol to prevent reduction of type II collagen, a primary component of articular cartilage, in chondrogenic ATDC5 cells. These findings agree with the results of a recent   study demonstrating the ability of paeonol to inhibit expression of MMPs and loss of type II collagen in rabbit chondrocytes. Also in accordance with the findings of the same study, we reveal that paeonol treatment inhibited activation of NF-jB [39]. Activation of NF-jB triggered by IjBa phosphorylation and subsequent nuclear translocation of p65 governs the expression of a massive array of inflammatory processes. Thus, blockade of NF-jB signalling is often cited as a valuable therapeutic target. Importantly, we found that treatment with paeonol could inhibit activation of NF-jB by significantly inhibiting IjBa and nuclear translocation of p65 protein induced by IL-1b.
In summary, the results of this study are among the first to demonstrate the potential of paeonol as an effective therapeutic agent against the pathogenesis of OA by virtue of its ability to downregulate expression of proinflammatory cytokines, chemokines, cellular adhesion molecules, degradative enzymes and to inhibit the IjBa/NF-jB signalling pathway.