Paeonol Ameliorates Inammation and Cartilage of Chondrocytes in Osteoarthritis by Upregulating SIRT1

Objective: To explore the possible role of paeonol on chondrocyte inammation and cartilage protection in osteoarthritis (OA). Methods: Primary chondrocytes were isolated from rat stie joints, and were identied through toluidine blue staining and immunouorescence staining of type II collagen. The chondrocytes were transfected with sh-SIRT1 or/and paeonol (0, 20, 50, 100, 200, 1000 mg/L) before OA modeling induced by IL-1β. ELISA determined the expressions of TNF-α, IL-17 and IL-6, and apoptotic rate was examined by ow cytometry. qRT-PCR and Western blot quantied the expressions of MMP-1, MMP-3, MMP-13, TIMP-1, cleaved-caspase-3, Bax, Bcl-2, and the proteins related to NF-κB pathway. Results: Increases in TNF-α, IL-17, IL-6, MMP-1, MMP-3 and MMP-13 and decrease in TIMP-1 were found in IL-1β stimulated chondrocytes. The apoptotic rate as well as the expressions of cleaved-caspase-3 and Bax was up-regulated, and Bcl-2 expression was suppressed in response to IL-1β treatment. NF-κB pathway was activated in IL-1β-stimulated chondrocytes. Paeonol enhanced SIRT1 expression to inactivate NF-κB pathway, thus ameliorating the secretion of inammatory cytokines, extracellular matrix degradation and chondrocyte apoptosis. Conclusion: Paeonol inhibits IL-1β induced inammation and extracellular matrix degradation in chondrocytes through up-regulating SIRT1 and suppressing NF-κB pathway.


Introduction
Osteoarthritis (OA), a chronic disease which impacts the lives of millions of people worldwide, primarily induces disability, joint stiffness, and pain [1]. The pathogenesis of OA, featured by extracellular matrix (ECM) degradation and cell stress, is primarily induced by micro-and macro-injuries that activate maladaptive repair responses, such as pro-in ammatory pathways of innate immunity [2]. Chondrocytes synthesize and secrete components of the ECM infrastructure as well as various enzymes responsible for matrix-degradation, aggrecan-degradation and hydrolysis, which consequently facilitates the degradation and elimination of denatured and dysfunctional ECM proteins, thus altering ECM structure [3]. IL-1β is generally applied for OA modeling for it can elicit matrix metalloproteinases (MMPs) and nitric oxide (NO) in chondrocytes, among which MMP-3 and MMP-13 act as the two most important collagenases involved in the degradation of the cartilage matrix in OA [4]. Tissue inhibitors of metalloproteinases (TIMPs), including TIMP-1 and TIMP-2, were generated to neutralize the physiological activities of MMPs [5]. The existing therapy of OA includes interventions that relief the symptoms only, and the only de nite treatment for OA is joint arthroplasty, which is expensive and requires revision in 10 ~ 15 years [6]. Inhibition of chondrocyte in ammation and ECM degradation could be a promising strategy to blunt OA progression.
Paeonol is the mainly effective compound in Paeonia lacti ora Pallas, Cynanchum paniculatum, and Paeonia suffruticosa Andr [7]. Its protective role in hepatocytes has been documented against oxidative stress and in ammation, and hepatocyte apoptosis in LPS/d-GalN-induced acute liver failure (ALF) in mice with both NF-κB and MARK signaling pathways involved [8]. More importantly, paeonol has been demonstrated to inhibit TNF-α and IL-6 expressions released in chondrocytes after IL-1β treatment [7], whereas, the precise mechanism has yet to be developed. SIRT1 has been denoted to be involved in the development of OA [9][10][11]. In addition, SIRT expression was decreased in degenerated cartilage, and SIRT1 inhibition in chondrocytes elicits hypertrophy and cartilage matrix degradation [12]. More importantly, paeonol up-regulated expression and nucleus accumulation of SIRT1 in high-glucoseinduced glomerular mesangial cells (GMCs) [13]. So far, it remains much to be seen how paeonol regulates OA development and whether it implicates in OA through mediating SIRT1.
In the present study, we examined the effects of paeonol on in ammation and ECM degradation in IL-1βstimulated chondrocytes from rat joints. Moreover, we analyzed the precise mechanism underlying the regulation of paeonol on OA is associated with SIRT1 and NF-κB signaling pathway.

Materials And Methods
Isolation and culture of chondrocytes Speci c pathogen free (SPF) Wistar rats (n = 5), weighting 180 ~ 200 g, were obtained from Hunan SJA Laboratory Animal Co., Ltd (Changsha, China). The rats were anesthetized with 50 mg/kg of pentobarbital sodium and then killed by cervical dislocation. The mice were immersed in 75% ethanol with their bilateral sti e joints collected under sterilized conditions. The articular cartilage was isolated and cut into pieces (1 mm × 1 mm × 1 mm), followed by washing with PBS. The pieces were then centrifuged at 800 r/min for 5 min prior to digestion by 0.25% pancreatin at 37℃ for 1 h. After the supernatant was removed, the cartilage was digested by 0.04% type II collagenase containing 5% FBS at 37℃ overnight using water bath. Then the cartilage was ltered using a 200 mesh screen before centrifuged at 800 r/min for 5 min. After the supernatant was discarded, the cells were washed and then were incubated with DMEM/F12 (Gibco, Grand Island, NY, USA) containing 10% FBS and 1% doubleantibody at 37℃ under 5% CO 2 . All experiments were in accordance with the guidance for the care and use of laboratory animals.

Identi cation of chondrocytes
When the cells grew over 80% of the slides, the cells were washed with PBS for twice and xed with 4% paraformaldehyde for 20 min at room temperature. Then toluidine blue staining and immuno uorescence staining of type II collagen were performed for identi cation of chondrocytes. Toluidine blue staining: The cells were subjected to staining by 1% toluidine blue for 30 min, dehydration by gradient alcohol and sealing by neutral balsam before observation under a microscope. Immuno uorescence staining: The cells were successively incubated with 3% H 2 O 2 for 10 min and with goat serum at room temperature for 15 min. COL2A1 antibody (sc-52658, 1:100, Santa Cruz, CA, USA) was added for incubation at 4℃ overnight, and then FITC-labeled secondary antibody was incubated with the chondrocytes for 1 h. The excessive secondary antibody was removed by PBS wash, after which uorescent quencher was added.
The chondrocytes were visualized and photographed under a uorescence microscope.

Treatment of chondrocytes
The powder of paeonol (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was dissolved in DMSO to prepare for 1.0 g/L mother solution. The mother solution was maintained at room temperature and was diluted into the appropriate concentration before the performance of following experiments. Paeonol (0, 20, 50, 100, 200 and 1,000 mg/L) was used to treat the chondrocytes for 24 h before the toxicity of chondrocytes was measured by MTT. Then the PBS-dissolved IL-1β (10 ng/ml, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) reagent was applied to induce OA model in chondrocytes for 24 h.

MTT
The chondrocytes were inoculated in 96-well plates (3,000 cells/well), and paeonol (0, 20, 50, 100, 200 and 1,000 mg/L) was added into each well. Each group has three repeated wells. The chondrocytes were cultured in an incubator at 37℃ under 5% CO 2 for 24 h, followed by incubation with MTT (5 mg/ml) dissolved by 10 µl of DMSO for 4 h. Then the OD value was examined at 570 nm.

Flow cytometry
After corresponding treatment, the concentration of chondrocytes in all groups were adjusted to 10 5 cell/ml. Suspension (3 ml) of each sample was collected in centrifuge tubes (10 ml) and centrifuged at 500 r/min for 5 min, after which the culture solution was removed. Then the samples were washed with PBS and centrifuged at 500 r/min for 5 min. After that, the supernatant was discarded, and the cells were re-suspended using 100 µl of binding buffer. Annexin V-FITC (5 µl) and PI (5 µl) were mixed and incubated with the cells in dark for 15 min. The uorescence of FITC and PI was detected by ow cytometer to analyze the apoptotic rate of chondrocytes. The detection on each group was performed in three times.

ELASA
The expressions of TNF-α, IL-17 and IL-6 were detected using ELISA kit (R&D Systems, MN, USA), and all procedures were measured according to the protocols.
qRT-PCR TRIZOL was applied to extract the total RNAs (Invitrogen, Carlsbad, CA, USA), and RNAs were reversely transcribed using reverse transcription kit (TaKaRa, Tokyo, Japan) according to the instruction. The expressions of RNAs were examined using LightCycler 480 (Roche, Indianapolis, IN, USA) and the reaction conditions were performed in accordance with the directions of uorescence quantitative PCR kit (SYBR Green Mix, Roche Diagnostics, Indianapolis, IN). The thermal cycle parameters were as follows: 95℃ for 5 s; 95℃ for 5 s, 60℃ for 10 s, 72℃ for 10 s (total of 45 cycles); and nally extension at 72℃ for 5 min. Each reaction was performed in triplication. GAPDH was used for normalization. Data were analyzed using 2 −ΔΔCt and ΔΔCt was expressed as (Ct target gene -Ct internal control ) experimental group -(Ct target gene -Ct internal control ) control group . The primers of all genes and their internal controls were shown in Table 1. Table 1 Primer sequences of all genes.

Statistical analysis
GraphPad prism7 was applied for statistical analysis, and all data were represented as average ± standard deviation (average ± SD). Difference between two groups was assessed through T test, and comparisons among multiple groups were measured using One-way analysis of variance with Dunnett's multiple comparisons test as post hoc test. P values of less than 0.5 were regarded as statistical signi cant.

Identi cation of primary chondrocytes
The extracted cells were subjected to toluidine blue staining and immuno uorescence staining of type II collagen. Toluidine blue staining suggested that the extracted cells were blue-violet (Fig. 1A), implying the generation of aggrecan in these cells. According to immuno uorescence staining of type II collagen, the extracted cells were full with green uorescence-represented type II collagen in the cytoplasm (Fig. 1B). All these results indicated that the extracted cells were chondrocytes.

Effect of paeonol on cartilage and in ammation in IL-1βstimulated chondrocytes
Paeonol (0, 20, 50, 100, 200, 1000 mg/L) was used to treat chondrocytes for 24 h before cell viability was detected by MTT assay. The cell toxicity experiments disclosed that paeonol (concentration less than 200 mg/L) had no toxic effect on chondrocytes, and chondrocyte viability was signi cantly decreased when chondrocytes were treated with paeonol at the concentration of 1,000 mg/L ( Fig. 2A, P < 0.05).
After pre-treatment of paeonol (0, 20, 50, 100, 200 mg/L) for 24 h, the chondrocytes were stimulated by 10 ng/ml of IL-1β for 24 h. In IL-1β group, the levels of TNF-α, IL-17 and IL-6 were notably elevated when compared with Control group (Fig. 2B, P < 0.05). Compared with IL-1β group, the expressions of above in ammatory cytokines were inhibited by paeonol pre-treatment in a dose dependent manner (Fig. 2B, P < 0.05). Flow cytometry uncovered that apoptotic rate in IL-1β group was marked higher than that in Control group, while paeonol treatment decreased apoptotic rate of chondrocytes in a dose dependent manner (Fig. 2E, P < 0.05). In addition, the expressions of cleaved-caspase-3 and Bax in IL-1β group were much higher than those in Control group, and Bcl-2 expression was downregulated in IL-1β group, compared with Control group (Fig. 2F, P < 0.05). Paeonol acted as a potent inhibitor of cleaved-caspase-3 and Bax, and upregulated Bcl-2 expression in a dose dependent manner (Fig. 2F, P < 0.05).

qRT-PCR and Western blot unraveled increases in
Aforementioned results con rmed that IL-1β treatment could activate in ammation in chondrocytes, and encourage degradation of extracellular matrix and chondrocyte apoptosis. More importantly, paeonol could impair IL-1β-triggered in ammation, extracellular matrix degradation and chondrocyte apoptosis in a dose dependent manner.

Paeonol regulates in ammation in chondrocytes and protects cartilage through SIRT1
The expressions of SIRT1 in chondrocytes treated with paeonol (0, 20, 50, 100, 200 mg/L) and IL-1β (10 ng/ml) were assessed by qRT-PCR and Western blot. In IL-1β group, chondrocytes had lower expression of SIRT1 than in Control group, and paeonol rescued SIRT1 expression in a dose dependent manner ( Fig. 3A-B, P < 0.05), suggesting the important role of SIRT1 in paeonol regulating chondrocyte in ammation and extracellular matrix degradation.
Then the chondrocytes were transfected with sh-SIRT1 or sh-NC before paeonol (200 mg/L) and IL-1β treatment. qRT-PCR and Western blot suggested the markedly decreased SIRT1 expression in chondrocytes after sh-SIRT1, compared with sh-NC group (Fig. 3C-D, P < 0.05).
After transfection of sh-SIRT1, the expressions of TNF-α, IL-17 and IL-6 was higher than those in sh-NC + Paeonol group (Fig. 3E, P < 0.05). In sh-SIRT1 + Paeonol group, the levels of MMP-1, MMP-3 and MMP-13 were increased in chondrocyte, and TIMP-1 expression was decreased when compared with sh-NC + Paeonol group (Fig. 3F-G, P < 0.05). Transfection of sh-SIRT1 strengthened the apoptotic rate of chondrocytes, expressions of cleaved-caspase-3 and Bax in addition to suppressing Bcl-2 expression, compared with sh-NC + Paeonol group (Fig. 3I, P < 0.05). Taken together, transfection of sh-SIRT1 could partially abolish the effect of paeonol on in ammation in chondrocytes and degradation of extracellular matrix, and paeonol acted its protective effects on chondrocytes through upregulating SIRT1.

Paeonol inhibits NF-κB signaling pathway
Compared with Control group, the expressions of p-IκBa and p-p65 were increased in IL-1β group, but downregulated by paeonol in a dose dependent manner in comparison to IL-1β group (Fig. 4A-B, P < 0.05).
Moreover, transfection of sh-SIRT1 signi cantly increased the expressions of p-IκBa and p-p65 in chondrocytes, compared with sh-NC-Paeonol group (Fig. 4A-B, P < 0.05). These results elucidated that IL-1β treatment could activate NF-κB signaling pathway in chondrocytes, and paeonol could inhibit the activation of NF-κB signaling pathway through upregulating SIRT1 in a dose dependent manner.

Discussion
OA is a multifactorial disorder characterized by low-grade, chronic in ammatory response, resulting in interactions between immune system and factors including local tissue damage and metabolic dysfunction [14]. Therefore, how to inhibit the release of in ammatory cytokines and block cellular signaling pathways is regarded as an attractive option for the management and treatment of OA. Collected evidence in present study supported that paeonol alleviated chondrocyte in ammation and ECM degradation induced by IL-1βvia enhancing SIRT1 expression. Our results indicated that NF-κB signaling pathway is activated in in ammatory chondrocytes, while whose activation can be suppressed by paeonol through regulating SIRT1.
Paeonol is the main component isolated from the root bark of paeonia suffruticosa, which has been reported to have pharmacological effects on in ammation and pain-related indication in diseases including OA [15]. However, little is known regarding the precise mechanism underlying the antiin ammatory effect of paeonol on OA. Here, chondrocytes from the sti e joint of rats were isolated and stimulated with IL-1β to explore the potential effects and mechanism of paeonol treatment in OA. We con rmed paeonol has no toxicity on chondrocytes through MTT assay, which showed that paeonol (less than 200 mg/L) had little toxicity on chondrocytes. Additionally, the expressions of TNF-α, IL-6 and IL-17 were markedly increased in IL-1β-treated chondrocytes. The in ammatory mediators such as IL-1β trigger the expressions of in ammatory factors such as TNF-α, leading to the enhanced secretion of IL-6 and IL-17 [16]. Moreover, paeonol treatment could suppress the expressions of in ammatory cytokines in a dose dependent manner. MMP-3 can cleave multiple ECM including aggrecan [17]. Both MMP-3 and MMP-13 are responsible for the digestion of type II collagen by causing the triple helix to unwind and inducing cleavage at the P4-P11' site [18]. In OA, the increased expressions of MMP-1, MMP-3, MMP-13 and decreased expression of TIMP-1 was found [19]. Consistently, the expressions of MMP-1, MMP-3 and MMP-13 were facilitated, and the expression of TIMP-1 was inhibited in IL-1β-stimulated chondrocytes. After paeonol treatment, MMP-1, MMP-3 and MMP-13 expressions were attenuated and TIMP-1 expression was elevated. In addition, the apoptotic rate of chondrocytes was increased by IL-1β stimulation and ameliorated by paeonol treatment. Taken together, paeonol is able to protect chondrocytes against in ammation, ECM degradation and cell apoptosis.
SIRT has been elucidated to exert its anti-in ammatory effect and regulate ECM in OA [20], and overexpression of SIRT1 in human chondrocytes leads to repression of MMP-3, MMP-8 and MMP-13 expressions [21]. Herein, we found that SIRT1 expression was hampered in the chondrocytes treated with IL-1β, while paeonol treatment could dose-dependently increase SIRT expression. Silence of SIRT1 enhanced the chondrocyte in ammation, apoptosis and ECM degradation, and the positive effects of paeonol were attenuated by SIRT1 silence. Therefore, we concluded that paeonol upregulated SIRT to implicate in OA development. A former study demonstrated that NF-κB signaling pathway has been activated in OA [22]. In monosodium urate (MSU)-induced arthritis (MIA), paeonol has been reported to reduce the expressions of TNF-α, IL-1β and IL-6 through inhibiting NF-κB-mediated proin ammatory cytokine production [23]. The implication of NF-κB signaling pathway in paeonol attenuating in ammatory response and apoptosis in IL-1β-stimulated chondrocytes was explored. In our study, NF-κB signaling pathway was activated by IL-1β in chondrocytes, and paeonol treatment reduced the expressions of proteins related to NF-κB signaling pathway. However, the inhibition of paeonol on NF-κB signaling pathway was neutralized by SIRT1 silence. P65, an indicator of NF-κB signaling pathway exists in the cytoplasm, and when activated by in ammatory cytokines such as IL-1β, p65 is phosphorylated and translocates into the nucleus, further leading to increases in the expressions of multiple in ammation-related genes like MMPs and IL-6 [24,25]. Interestingly, paeonol has been demonstrated to inhibit the phosphorylation of p65, thus inactivating NF-κB signaling pathway [26]. Accordingly, this study declared that paeonol dose-dependently enhanced SIRT1 expression to inactivate NF-κB signaling pathway.
Our results con rm the potential of paeonol as a candidate for OA drugs by virtue of its ability to suppress chondrocyte in ammation and ECM degradation through upregulating SIRT1 and inactivating NF-κB signaling pathway. Declarations