β-catenin signaling pathway

Biomedicine & Pharmacotherapy 122 (2020) 109708

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Jiawei Yanghe decoction ameliorates cartilage degradation in vitro and vivo via Wnt/β-catenin signaling pathway

T

Hanting Xiaa, Duanguang Caob, Fo Yangb, Wenlong Yanga, Wei Lib, Pu Liub, Shuhao Wangb, Fengyun Yangb,* a b

Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine, China Jiangxi University of Traditional Chinese Medicine, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Traditional Chinese medicine Wnt/β-catenin signaling pathway Osteoarthritis Jiawei yanghe decoction

Jiawei Yanghe decoction (JWYHD) is a Traditional Chinese Medicine (TCM) formula for the treatment of osteoarthritis (OA), however the underlying mechanisms of action of JWYHD in OA are not fully explored. This study investigates how JWYHD protects cartilage from degradation via Wnt/β-catenin signaling pathway. The chondroprotective and anti-inflammatory effect of JWYHD on chondrocytes in vitro and on MIA-induced OA rat model in vivo were investigated. In vitro, JWYHD increased the chondrocyte viability against interleukin (IL)-1βinduced chondrocytes apoptosis and preserved glycosaminoglycans in the extracellular matrix. JWYHD promoted chondrocyte viability against apoptosis, decreased MMP-3, MMP-13, Caspase-3, Caspase-9 via Wnt/βcatenin signaling pathway in both IL-1β-induced and Licl-induced chondrocytes. The qRT-PCR and western blot results showed that mRNA and protein expressions of Wnt signaling pathway related genes β-catenin and CyclinD1, apoptosis related genes Casapase-3 and Caspase-9, collagen degradation related genes Metalloproteinase (MMP)-3 and MMP-13 were up-regulated, and Col2a1 was down-regulated on IL-1β-induced chondrocytes. Treatment with JWYHD reversed these effects in a dose-dependent manner. Licl was used as Wnt/ β-catenin signaling pathway activator in chondrocytes to determine the molecular mechanisms. Activation of Wnt signaling pathway by Licl up-regulated β-catenin, CyclinD1, Axin2, Caspase-3, Caspase-9, MMP-3, MMP-13 and IL-1β. These effects were blocked by JWYHD treatment. Furthermore, 75 Sprawl-Dawley rats were used to verify the results obtained in vitro. A total of 75 rats were randomly divided into the control group (no MIAinduced OA, received intragastric administration of an equivalent amount of saline), the OA group (MIA-induced OA, received intragastric administration of an equivalent amount of saline), and the JWYHD treatment group (MIA-induced OA, received intragastric administration of an equivalent amount of various concentrations of JWYHD at 1.4/2.7/5.5 g/kg). After 8 weeks of administration, all rats were sacrificed. JWYHD decreased the MIA-induced up-regulation of β-catenin, CyclinD1, Caspase-3, Caspase-9, MMP-3 and MMP-13 protein expressions in cartilage. It was also demonstrated that JWYHD decreased serum and synovium pro-inflammatory cytokines, IL-1β, IL-6 and TNF-α in MIA-induced OA rats and ameliorated the cartilage degradation. Histopathological staining, macroscopic observation and micro-CT scan with 3-dimension remodeling showed a cartilage protective effect of JWYHD. In conclusion, JWYHD possess multiple capabilities including preventing chondrocyte apoptosis, preserving integrity of extracellular matrix and anti-inflammatory effect in the treatment of OA both in vitro and in vivo.

1. Introduction Osteoarthritis (OA) is the most common degenerative arthritis which is characterized by the formation of osteophyte, articular cartilage degradation, subchondral sclerosis and synovitis [1]. Among the population of over 65 years, the incidence of knee OA is 50%, of which 8.1% is systematic knee OA [2], and is still on rise [2,3]. Systematic

⁎

knee OA is associated with increased cardiovascular incidences and allcause mortality [3–6]. Up to now, no specific treatment is available for OA. There is consensus that the development of pathological lesions due to cartilage degradation involves OA [7]. Cartilage degradation is characterized by chondrocyte apoptosis and degradation of extracellular matrix, and these overtime decreases the integrity of cartilage

Corresponding author. E-mail address: [email protected] (F. Yang).

https://doi.org/10.1016/j.biopha.2019.109708 Received 8 June 2019; Received in revised form 7 November 2019; Accepted 25 November 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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[8,9]. Chondrocyte is the only cell type in cartilage and chondrocytes apoptosis is directly involved in cartilage degradation [10]. Extracellular matrix is mainly composed of collagen, proteoglycans and hyaluronic acid. Chondrocytes contributes little to the physical function of cartilage directly, and their main function is secreting extracellular matrix [11]. There are multiple causes of extracellular matrix degradation, such as pro-inflammatory cytokines and metalloproteinases [12,13]. Metalloproteinase enzymes produced by articular chondrocytes, like matrix metalloproteinases MMP-3 and MMP-13 participate in OA progression by degrading extracellular matrix and result in degradation of cartilage [14,15]. Overproduction of pro-inflammatory cytokines of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are reported to be involved in the pathogenesis of OA directly [13]. Pro-inflammatory cytokines induce multiple enzymes including MMPs resulting in cartilage degradation [16]. Wnt/β-catenin signaling pathway was found to play a crucial part in aggravating OA by promoting overproduction of MMPs [17]. Additionally, Wnt/β-catenin signaling pathway regulates the maturation, differentiation and apoptosis of chondrocytes [18]. The activation of canonical Wnt signaling pathway has been elucidated [19]. Briefly, Axin, glycogen synthase kinase 3β (GSK-3β), β-catenin consist the βcatenin destruction complex in the microenvironment without Wnt or other activators. The complex recycles by phosphorylation, ubiquitination and degradation of β-catenin. Signaling activators, including Wnt, Licl or other activators, lead to the binding of β-catenin degradation complex and LDL receptor related protein(LRP). Binding to LRP suppresses the ubiquitination and degradation of β-catenin. Therefore, newly synthesized β-catenin translocate and accumulates in the nucleus, thereby activating transcription of targeted genes of canonical Wnt/β-catenin signaling pathway including MMPs [20]. Reduced production of MMPs by blocking canonical Wnt signaling pathway leads to amelioration of OA in animal [21]. Additionally, results showed that Licl-induced chondrocytes resulted in up-regulation of IL-1β, which indicates the possible connection between pro-inflammatory cytokines and Wnt/β-catenin signaling pathway. Currently, treatment of OA, especially with non-steroid anti-inflammatory drugs, has not been effective and is associated with undesirable adverse outcomes such as gastrointestinal hemorrhage and cardiovascular defects. Thus, natural production, including traditional Chinese medicine (TCM), has attracted much attention. TCM has a long history in the treatment of OA. Jiawei Yanghe decoction (JWYHD) is a TCM formula that is widely used to treat OA. Previous studies showed the cartilage protective effect of JWYHD [22]. However, the effects of JWYHD on the chondrocytes and the precise mechanisms are not well understood. In this study, chondroprotective and anti-inflammatory effect of JWYHD on chondrocytes and on MIA-induced OA rat model are investigated. It was found that JWYHD ameliorated cartilage from degradation by promoting proliferation of chondrocytes and preserving the integrity of extracellular matrix, which is correlated with downregulation of pro-inflammatory cytokines and metalloproteinase by suppressing Wnt/β-catenin signaling pathway.

Table 1 Composition of Jiawei Yanghe Decoction (JWYHD). Herb name

Relative proportion

Radix rehmanniae rraeparata Cinnamomum cassia Ephdra sinica stapf Colla Cornus Cervi Fructus chaenomelis Lagenariae Baked ginger Semen Brassicae Lignum millettiae stephania tetrandra radix liquiritiae

15 10 10 10 8 6 10 20 10 3

PrimeScript RT Reagent kit was procured from TaKaRa (Dalian, China). Primer synthesis was performed by Shanghai Sangon Biotech (Shanghai, China). Primary antibodies against CyclinD1, Caspase-3, Caspase-9, MMP-3, MMP-13, Axin2 and Col2a1 were obtained from Abcam (Cambridge, USA). β-catenin and β-actin primary antibodies were purchased from Proteintech (Chicago, USA). Secondary antibody was purchased from Proteintech. No-fat milk was purchased from Dickinson and Company (Difco, USA). IL-1β, IL-6 and TNF-α Elisa kit were purchased from Antibody-Sunlong Biotech (Hangzhou, China). JWYHD is composed of Shu Di Huang (Radix rehmanniae rraeparata), Rou Gui (Cinnamomum cassia), Ma Huang (Ephdra sinica stapf), Lu Jiao Jiao (Colla Cornus cervi), Mu Gua (Fructus chaenomelis lagenariae), Pao Jiang (Baked ginger), Bai Jie Zi (Semen brassicae), Ji Xue Teng (Lignum millettiae), Fang Ji (stephania tetrandra), Gan Cao (radix liquiritiae). All herbs were purchased from the Dispensary of Traditional Chinese hospital of Jiangxi Province. 2.2. Preparation of JWYHD Herbs used to prepare JWYHD were formulated according to the relative proportions as shown in Table 1. All original decoction was mixed and soaked in 4 times volumes of water, boiled twice for 30 min, extracted 3 times, concentrated and dried to form a powder. Dry decoction 26.3 g was prepared per 100 g original herbs. Dry extraction was achieved using DMSO at a concentration of 1 g/ml and stored at -20 ℃ 2.3. Chemical components and quality control of JWYHD To determine the main chemical components of JWYHD, HPLC-MS analysis was conducted. 200 mg dry JWYHD decoction was added with 1 ml methanol, whirled for 10 min, centrifuged at 4 ℃ for 10 min. Centrifugal force was set to be 20,000 xg. Supernatant was collected and filtered with 0.22 μm filter membrane. Resultant sample was subjected to chromatographic analysis on an Ultimate 3000 RS system (Thermo Fisher Scientific, MA, USA) equipped with a Thermo Hypersil GOLD column (φ 2.1 × 100 mm, 1.9 μm) and the MS spectra were acquired by an Q Executive high-resolution mass spectrometer (Thermo Fisher Scientific, MA, USA). The mobile phases were (A) 0.1% formic acid in water (B) and 0.1% formic acid in acetonitrile, and the gradient elution program was (time/ B%): 0−1 min, 2%; 1−5 min, 2%–20%; 5−10 min, 20–50%; 10−15 min, 50–80%; 15–20 min, 80–95%; 20−25 min, 95%; 26–30 min, 2%. The chromatographic analysis was performed at 35℃ with a flow rate 0.3 ml/min and injection volume 15 μL. The mass spectrometer parameters were as follows: spary voltage was set at 3.8 kV at positive mode. Capillary temperature was set at 300℃. Argon was used as collision gas. Nitrogen was used as sheath gas and aux gas. Aux gas heater temperature was set to be 350℃. Resultant data was analyzed by CD 2.1 software (Thermo Fisher Scientific, MA, USA) and then compared with online databases.

2. Materials and methods 2.1. Materials and reagents Monoiodoacetic acid (MIA) was purchased from Macklin. IL-1β, TNF-α, Dickkopf-1(DKK-1) were purchased from Peprotech (Rocky Hill, NJ, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). Penicillin, streptomycin and 0.25% trypsin, collagenase II,3-(4,5dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO) were purchased from Solarbio (Beijing, China). TRIzol reagent was provided by Invitrogen (Carlsbad, USA). 2

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2.9. Animal study

Furthermore, for the reproducibility of this study, 2 main chemical components of JWYHD, calycosin and sinapine thiocyanate was selected to be quantified and used as landmarks of quality control of JWYHD. The peak areas of calycosin and sinapine thiocyanate were taken as the longitudinal coordinate and the densities of them was taken as the transverse coordinate, separately. The standard cure was obtained by regression with the weighted coefficient (1/x2).

Articular cartilage specimens were obtained from knee joint of 2week-old rats housed in the Laboratory Animal Science and Technology Center of Jiangxi University of Traditional Chinese Medicine (Nanchang City, Jiangxi Province, China). Specimens were cut into pieces and then digested with 0.25% trypsin-EDTA solution for 1 h followed by 0.1% collagenase Ⅱ for 4 h to obtain chondrocytes. Chondrocytes were seeded in 25 cm2 culture flasks and cultured with DMEM containing 10% FBS and antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin) in 5% CO2 at 37 ℃ conditions. Designated as Passage 0 (P0). Chondrocytes were passaged at a ratio of 1:3 to reach a confluence of 80%, designated as P0, P1, P2 and P3. P3 chondrocytes were used for the subsequent experiments. Chondrocytes were identified with toluidine blue staining as previously described [23].

Seventy-five female Sprawl-Dawley rats (200 ± 20 g) were purchased from Laboratory Animal Science and Technology Center of Jiangxi University of Traditional Chinese Medicine (Nanchang City, Jiangxi Province, China). All rats were raised in a pathogen free (SPF) environment. All rats were allowed free access to diet and water. All animal care and protocols were approved by the Committee of Management and use of Laboratory Animals of Jiangxi University of Traditional Chinese Medicine (Nanchang City, Jiangxi Province, China). All animal experiments complied with the Guide for the National Institutes of Health guide for the care and use of Laboratory animals. MIA-induced OA rat models were performed as previously described [24]. Briefly, OA rats received an intra-articular injection of 3 mg MIA diluted in 50 μl saline. Subsequently, all rats were randomly divided into 5 groups: the control group (injected with saline instead of MIA solution; treated with 2 ml saline once a day), the model group (MIAinduced OA; treated with 2 ml saline once a day), the JWYHD groups (MIA-induced OA; treated with 1.4/2.7/5.5 g/kg JWYHD). Treatment was given once a day by gavage for 8 weeks. After treatment, all rats were sacrificed, synovium and knee joints were obtained for further analysis.

2.5. Cell treatment

2.10. Arthritis index

P3 chondrocytes were seeded in 6-well plates at an initial density of 5 × 105 cells/well and then serum starved for 12 h. Chondrocytes were pretreated with a series of JWYHD concentrations (50, 100, 200, 400 μg/ml) for 24 h with or without 10 ng/ml IL-1β for a further 12 h. Alternatively, P3 chondrocytes were incubated with 400 μg/ml JWYHD or 10 ng/ml DKK-1 with or without 20 mM Licl.

Arthritis index was introduced in this study. A total number of 5 time points (0, 2, 4, 6, 8 weeks) was set to measure the arthritis index. The arthritis index was measured by redness and swelling and classified into 5 different degree. 0 ° : no red spots or joint swelling; 1 °: little red spots or mild joint swelling; 2 degrees: moderate joint redness and swelling; 3 degrees: severe redness and swelling of the joint; 4 degrees: severely redness, swelling and incapacity of weight bearing of the joint.

2.4. Cell isolation and culture

2.6. MTT assay 2.11. Histological analysis and macroscopic observation P3 chondrocytes were cultured in 96-well plates at about 5 × 103 cells in each well. Chondrocytes were serum-starved for 12 h followed by treatment with various concentration of JWYHD (10, 50, 100, 200, 400, 800, 1000 μg/ml) for various time periods (24, 48, 72 h), respectively. Six duplications were applied for each well. Alternatively, chondrocytes were pre-treated with JWYHD, and then treated with 10 ng/ml IL-1β. Six wells without JWYHD and another six wells without cells were used as the control and blank, respectively. 5 mg/ml MTT were mixed with the culture medium at a concentration of 20 μl for each well at 37 ℃ for 4 h. The supernatant was removed and then 150 μl DMSO was added per well. Optical density was measured at 570 nm with a multimode reader (Spark 10 M, TECAN, Switzerland).

Samples were fixed in 4% paraformaldehyde for 24 h. They were then fixed and decalcified in 15% EDTA for 21 days. The samples were dehydrated with a series of ethanol concentration. Transparent samples were obtained by soaking in Xylene for 45 min. They were then immersed and embedded in paraffin blocks before they were sliced into 5 μm sections. H.E staining and Safranning O staining were performed as previously described [25]. Modified Mankin scoring was performed by two independent researchers. Macroscopic observation of femoral condyle was also performed. 2.12. Micro-CT scan Micro-CT scan (Skyscan 1176, Bruker micro CT N.V, Kontich, Belgium) and 3-dimension remodeling was applied for radiographic observation. The left knee was harvested and cut to fit the appropriate size for micro-CT scan. Percentage of trabecular bone volume fraction (BV/TV) was performed.

2.7. DAPI staining Chondrocytes were fixed with paraformaldehyde and then blocked with goat serum. Then stained by incubating with DAPI for 5 min. The slide was visualized with a fluorescence microscope and quantified with Image-Pro Plus 6.0 image analysis software.

2.13. Measurement of pro-inflammatory cytokines vivo 2.8. Alcian blue staining of glycosaminoglycans Rat plasma was collected through orbital venous plexus and then centrifuged to obtain serum samples. Rats were sacrificed and the synovium was resected and then homogenized with PBS. The supernatant liquid was collected after centrifugation. These samples were used for ELISA assay for IL-1β, IL-6 and TNF-α according to the manufacturer’s instructions.

P3 chondrocytes were incubated in 24-well plates. The initial density was set to be 5 × 104 per well. Chondrocytes were pre-treated with various concentrations of JWYHD for 24 h followed by 10 ng/ml IL-1βstimulation for 12 h. After treatment, the chondrocytes were washed three times. Subsequently, chondrocytes were fixed in methanol for 20 min at -20 ℃. 0.5% alcian dissolved in 1 N HCL was used to stain the fixed chondrocytes overnight at room temperature. For further analysis, the stained cells were lysed followed by measurement of optical density at 630 nm with a multimode reader (Spark 10 M, TECAN, Switzerland).

2.14. Western blot Total protein samples were extracted using protein extraction kit 3

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Table 2 Primer sequence. Gene

Forward

Reverse

CyclinD1 β-catenin Caspase-3 Caspase-9 MMP-3 MMP-13 Col2a1 18S rRNA

5′-TCAAGTGTGACCCGGACTG-3' 5′-GCGACTAAGCAGGAAGGGAT-3' 5′GGAGCTTGGAACGCGAAGAA-3' 5′-GATATTCAGCGGGCAGGCTC-3' 5′-CAATCCCTCTATGGACCTCCC-3' 5′-CAAGCAGCTCCAAAGGCTAC-3' 5′-GCCAGGATGCCCGAAAATTA-3' 5′-TGGTTGATCCTGCCAGT-3'

5′GACCAGCTTCTTCCTCCACTT-3' 5′-CCCACTTGGCACACCATCAT-3' 5′ACACAAGCCCATTTCAGGGT-3' 5′-GCAGGAGATGAAGCGAGGAA-3' 5′-GGTCCTGAGAGATTTTCGCCA-3' 5′-CCTTGGAGACTTTGGTGAATGT -3' 5′-GTCACCTCTGGGTCCTTGTTC-3' 5′-TGATCCTTCTGCAGGTTCACC-3'

components were oleanolic acid, ephedrine hydrochloride, sinapine thiocyanate, liquiritin, trans cinnamaldehyde, calycosin and 6-gingerol. Furthermore, by comparing to the respective standards, contents of the 7 main chemical components were analyzed and the results were shown in Table 3.

following the manufacturer’s instructions. BCA Assay kit was used to measure the concentration of extracted protein. Protein samples were separated by sulphate polyacrylamide gel electrophoresis and then blotted onto polyvinylidene fluoride membranes. The proteins on the blot were assessed using primary antibodies against β-catenin, CyclinD1, Caspase-3, Caspase-9, MMP-3, MMP-13, Col2a1, Axin2 and β-actin followed by HRP-conjugated goat anti-mouse antibody or antirabbit secondary antibody. Blots were visualized using the electrochemiluminescence method. The density of protein bands was quantified with Image Lab software.

3.2. Identification of cultured chondrocytes Isolated chondrocytes were passaged to passage 3 (P3). Identification of P1 to P3 chondrocytes was achieved by toluidine blue staining. As shown in Fig.2E-G, blue stained area demonstrates rich existence of extracellular matrix of glycosaminoglycans. Cultured chondrocyte, P1 to P3, possessed typical characteristics of chondrocytes as previous described. Results showed that cultured chondrocytes were appropriate for sequence experiments.

2.15. Quantitative real-time polymerase chain reaction (qRT-PCR) Total cellular RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, USA) according to the manufacture’s instruction. Complementary DNA (cDNA) was reverse-transcribed with PrimeScript-RT reagent kit (TaKaRa Biotechnology Co, Ltd., Japan). The mRNA levels of β-catenin, CyclinD1, Caspase-3, Caspase-9, MMP-3, MMP-13, Col2a1 and 18 s rRNA were detected by quantitative real-time PCR with SYBR Premix Ex Taq kit (TaKaRa Biotechnology Co, Ltd., Japan). Primer sequences used are shown in Table 2 and the specificity of sequences was verified using the BLAST algorithm of National Center for Biotechnology Information. Data were normalized to 18 s rRNA and analyzed by 2(−ΔΔCT) method.

3.3. Effect of JWYHD on the viability of chondrocytes incubated with or without IL-1β The effect of JWYHD on proliferation of chondrocytes was assessed at various concentration of JWYHD (50, 100, 200, 400, 800, 1000 μg/ ml) pretreated chondrocytes followed by incubating with or without IL1β for different times. As shown in Fig.2A, no significant differences were found in chondrocytes incubated with 50, 100, 200, 400 μg/ml JWYHD for 24 h and 48 h (P > 0.05). Mild cytotoxicity effect was found in chondrocytes incubated with JWYHD at a concentration of 800 and 1000 μg/ml compared to the controls(*P < 0.05). Additionally, JWYHD treated chondrocytes more than 72 h had equal cytotoxicity compared to the control(*P < 0.05). Correspondingly, as illustrated in Fig.2B, proliferation of IL-1β-induced chondrocytes were reduced (#P < 0.05) and treatment of JWYHD reversed this trend at 50, 100, 200, 400 μg/ml concentrations in a dose-dependent manner compared to the IL-1β-induced chondrocytes (*P < 0.05). Thus, 10 ng/ml IL-1β and JWYHD at 50, 100, 200 400 μg/ml concentrations were applied in further experiments. Furthermore, DAPI staining was

3. Results 3.1. HPLC analysis and quality control of JWYHD As shown in Fig.1, 6 independent batches of JWYHD used in this study were analyzed by HPLC. Overlapping chromatograms showed in Fig.1 illustrated that the stability of JWYHD were confirmed. Thus, the reproductivity of the results showed in vitro and vivo experiments were considered to be sufficient. 7 main chemical components were identified by HPLC system and its finger prints. Identified chemical

Fig. 1. HPLC-,MS analysis results of JWYHD. 1: oleanolic acid, 2: ephedrine hydrochloride, 3: sinapine thiocyanate, 4: liquiritin, 5: trans cinnamaldehyde, 6: calycosin and 7: 6-gingerol. gradient elution program was (time/B%): 0−1 min, 2%; 1−5 min, 2 %–20 %; 5−10 min, 20–50 %; 10−15 min, 50–80 %; 15–20 min, 80–95 %; 20−25 min, 95%; 26–30 min, 2%.

4

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Table 3 Main Compounds of JWYHD. NO.

Compounds

tR/min

m/z

Chemical forumula

Content(ug/ml)

1 2 3 4 5 6 7

oleanolic acid Ephedrine Hydrochloride Sinapine thiocyanate Liquiritin Trans-Cinnamaldehyde Calycosin 6-Gingerol

1.55 5.95 7.18 8.92 10.62 11.06 12.06

259.0926 148.1117 251.0912 137.0229 105.0702 225.0544 259.2058

C30H48O3 C10H15NO*HCl C16H24NO5•SCN C21H22O9 C9H8O C16H12O5 C17H26O4

25.433 19.85 272.519 8.847 15.7 1.033 12.262

Fig. 2. Effect of JWYHD on the viability of chondrocytes incubated with or without IL-1β. Proliferation and viability of chondrocytes incubated with or without Interleukin (IL)-1β was increased by JWYHD. (A) Chondrocytes treated with various concentration of JWYHD (50, 100, 200, 400, 800, 1000 μg/ml) for various time (24, 48, 72 h). *P < 0.05 compared to the relative in the control. (B) Chondrocytes pre-treated with various concentration of JWYHD (50, 100, 200, 400 μg/ml) incubated with IL-1β for 24 h and 48 h. #P < 0.05 compared to the relative in the control. *P < 0.05 compared to the relative in the IL-1β-induced chondrocytes. (C) Chondrocytes pre-incubated with various concentrations (50, 100, 200, 400 μg/ml) of JWYHD followed by 10 ng/ml IL-1β-stimulation for 12 h. DAPI staining was performed to observe the apoptosis chondrocytes rate. (D) Percentage of apoptosis chondrocytes of various concentration of JWYHD. ##P < 0.01 compared to the control, *P < 0.05, **P < 0.01 compared to IL-1β-induced chondrocytes. (E–G) illustrated identification of cultured chondrocyte. Toluidine blue staining were performed as in previous study. (E) Passage 1, (F) Passage 2 and (G) Passage 3 cultured chondrocytes were stained with toluidine blue. (H) JWYHD preserved glycosaminoglycans in IL-1β-induced chondrocytes. 5

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cartilage tissue samples of MIA-induced OA rats were analyzed using Western blot. As shown in Fig.5, β-catenin, CyclinD1, Caspase-3, Caspase-9, MMP-3 and MMP-13 were significantly increased in MIA-induced OA rats, meanwhile the Col2a1 expression was significantly decreased compared to the control (#P < 0.05, ##P < 0.01). Treatment with JWYHD blocked these effects by MIA, similar to in vitro. Surprisingly, JWYHD possessed a dose-dependent manner in vitro but this was not reproduced in vivo. The effect of JWYHD seems not strong with the increasing dosage and the concentration of 2.7 g/kg has the most prominent effect among the various concentrations of JWYHD (1.4, 2.7, 5.5 g/kg).

performed to validate the MTT results shown in Fig.2B. Apoptotic chondrocytes possessed certain features, such as changes in morphology and brighter blue staining of the nucleus. As illustrated in Fig.2C, number of apoptosis chondrocytes were significantly increased by IL-1-stimulation. Treatment with JWYHD decreased the apoptosis of chondrocytes. Fig.2D demonstrated that JWYHD protected chondrocytes from IL-1-stimulated apoptosis with dose-dependent manner. JWYHD preserved glycosaminoglycans in IL-1β-induced chondrocytes Alcian Blue staining was conducted on chondrocytes to evaluate the effects of JWYHD on preservation of glycosaminoglycans. As shown in Fig.1H, IL-1β-induced loss of proteoglycans in chondrocytes compared to the control (##P < 0.01). Treatment of JWYHD reversed the trend in a dose-dependent manner compared to the IL-1β-induced chondrocytes (**P < 0.001). Results showed that JWYHD significantly reversed IL-1β-induced loss of glycosaminoglycans, hence protection effect on extracellular matrix.

3.7. JWYHD ameliorated arthritis index in MIA-induced OA rats The knee joint arthritis index of all rats was measured. As shown in Fig.6 D, arthritis index of the knee joint of OA rats was significantly increased compared to the sham group at 0, 2, 4, 6, 8 weeks(P < 0.05). Treatment of JWYHD significantly reversed the trend. At a dosage of 2.7 g/kg JWYHD possessed the most distinct effect among the 1.4 g/kg, 2.7 g/kg and 5.5 g/kg dosages. In line with the ELISA and staining tests in vivo, no dosage-dependent manner was found in arthritis index test and 5.5 g/kg JWYHD showed only minor effect and no significant difference was found on downregulating the arthritis index among all the time points. 1.4 g/kg JWYHD showed its effects from 4 weeks to 8 weeks, meanwhile the time point of 2.7 g/kg JWYHD was started from 2 weeks.

3.4. Inhibition of Wnt/β-catenin signaling pathway in Licl-induced chondrocytes by JWYHD To explore the specified Wnt/β-catenin signaling pathway inhibition effects of JWYHD, an activator Licl and an inhibitor DKK-1 were used. Wnt/β-catenin signaling pathway had effect on protein expressions of β-catenin and cyclinD1, pro-inflammatory protein IL-1β, cell apoptosis related proteins Caspase-3 and Caspase-9, metalloproteinase of MMP-3 and MMP-13 on Licl-induced chondrocytes (##P < 0.01) significantly increased. As shown in Fig.3, treatment with 400 μg/ml concentration of JWYHD reversed these trends on Licl-induced chondrocytes (*P < 0.05, **P < 0.01). JWYHD and DKK-1 suppressed the β-catenin, Axin2 and cyclinD1 on chondrocytes incubated with or without Licl compared to the comparative control (##P < 0.01, **P < 0.01). Statistical differences were found between JWYHD and DKK-1 treated chondrocytes (&&P < 0.01) and the effect of JWYHD was lower than DKK-1. Results show that activated Wnt/β-catenin signaling pathway resulted in up-regulation of pro-inflammatory, proapoptosis and pro-degradation activities in Licl-induced chondrocytes and JWYHD suppressed these trends by inhibiting Wnt/β-catenin signaling pathway.

3.8. JWYHD decreased serum and synovium pro-inflammatory cytokines of IL-1β, IL-6 and TNF-α in MIA-induced OA rats ELISA was conducted to evaluate whether JWYHD decreased proinflammatory cytokines of IL-1β, IL-6 and TNF-α in vivo. As shown in Fig.5I-N, serum and synovium IL-1β, IL-6 and TNF-α were significantly increased in MIA-induced OA rats cartilage compared to the control (#P < 0.05, ##P < 0.01) and intragastric administration of JWYHD for 8 weeks reversed these trends except in serum IL-6 compared to the MIA-induced group (*P < 0.05, **P < 0.01). Just like previous results, JWYHD did not increase with increasing dosage in vivo and a concentration of 2.7 g/kg was most effective. This result demonstrated that oral administration of JWYHD on MIA-induced OA rats had antiinflammatory effect.

3.5. Effect of JWYHD on protein and mRNA expressions of β-catenin, CyclinD1, Caspase-3, Caspase-9, MMP-3 and MMP-13 on IL-1β-induced chondrocytes

3.9. JWYHD ameliorated cartilage degradation in the MIA-induced OA rats β-catenin, CyclinD1, Caspase-3, Caspase-9, MMP-3, MMP-13 and Col2a1 protein and mRNA expressions were assessed by western blotting and qRT-PCR. As shown in Fig.4A-H, IL-1β-stimulation significantly increased protein expressions of β-catenin, CyclinD1, Caspase-3, Caspase-9, MMP-3 and MMP-13. Meanwhile Col2a1 was downregulated. Various concentrations of JWYHD (50, 100, 200, 400 μg/ml) possessed inhibition effects of these trends in a dose-dependent manner. Fig. 4I-O illustrated that mRNA expressions of β-catenin, CyclinD1, Caspase-3, Caspase-9, MMP-3, MMP-13 and Col2a1 varied with protein expressions. The results demonstrated that JWYHD down-regulated Wnt/β-catenin signaling pathway related genes of βcatenin and CyclinD1, apoptosis related genes of Caspase-3 and Caspase-9, metalloproteinase of MMP-3 and MMP-13. Meanwhile JWYHD up-regulated Col2a1, the most prominent part involved in the extracellular matrix of chondrocytes.

Multiple methods, including histopathologic observation, micro-CT scan with 3-dimension remodeling and macroscopic observation, were applied on MIA-induced OA rats to validate the anti-osteoarthritic effect of JWYHD in vivo, as shown in Fig.6A. Histopathological revealed that MIA-induced OA rats showed significant OA-like cartilage degradation features, including abraded surface, thinner layer and articular surface fibrillation. SeO staining results showed there was low Safranning O staining area in MIA-induced group, indicating that proteoglycan of ECM degraded. Correspondingly, Modified Mankin Scoring result indicated significantly higher MIA-induced group compared to the control, as shown in Fig.6B (#P < 0.05). The treatment of JWYHD reversed the histopathological changes induced by MIA. JWYHD treated OA group, especially at a concentration of 2.7 g/kg, showed smooth articular surface and seldom fast-green stained area, which indicates the normal function and integrity of cartilage and ECM. Micro-CT scan and 3-dimension remodeling results illustrated that significant destruction of the bone, formation of osteophyte and deviation of lower limb force line in MIA-induced OA rats were ameliorated by the treatment of JWYHD. BV/TV summarized the quantified results of micro-CT scan and 3-dimension remodeling, as shown in Fig.6C. Corelate with the Western blot results of in vivo mentioned above, JWYHD didn’t ameliorate MIA-induced cartilage degradation in a dose-

3.6. Effects of JWYHD on protein expressions of β-catenin, CyclinD1, Caspase-3, Caspase-9, MMP-3, MMP-13 and Col2a1 in MIA-induced OA rats To further confirm the above observations in in vitro experiments were performed to determine the protein expressions of β-catenin, CyclinD1, Caspase-3, Caspase-9, MMP-3, MMP-13 and Col2a1 in the 6

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Fig. 3. Inhibition of Wnt/β-catenin signaling pathway in vitro by JWYHD. (A) Licl-induced chondrocytes manifested up-regulation of β-catenin, CyclinD1, IL-1β, Caspase-3, Caspase-9, MMP-3 and MMP-13. Licl-induced up-regulation of protein expressions of (B) CyclinD1, (C) β-catenin, (D) IL-1β, (E) Caspase-3, (F) Caspase-9, (G) MMP-3 (H) MMP-13 and (I)Axin2 were reversed by treatment of various concentration of JWYHD (50, 100, 200, 400 μg/ml). ##P < 0.01 compared to the control group, **P < 0.01 compared to the Licl-induced group, &&P < 0.01 compared to relative treatment group.

BSHXD and DHJSD TCM formulas is JWYHD, which possess a long history and clinical effectiveness in the treatment of OA but has not been adequately studied. JWYHD originates from Yanghe Decoction in Qing dynasty. A famous Chinese medical doctor Wang Hongxu first recorded Yanghe decoction in his work, Life-saving manual of Diagnosis and Treatment of External Diseases (Waike Zhengzhi Quansheng Ji). Yanghe decoction possesses the function of warming Yang and nourishing blood, dispersing cold and activating stagnancy according to Chinese medicine theory. For hundreds of years, Yanghe decoction has proved to be useful in treatment of non-infectious inflammation including osteoarthritis. Yanghe decoction has been modified to enhance its effect on osteoarthritis of the knee, which is known as Jiawei Yanghe decoction. JWYHD consists of Rehmanniae Praeparata, Cinnamomum Cassia, Ephdra Sinica Stapf, Colla Cornus Cervi, Fructus Chaenomeles Lagenariae, baked ginger, Semen Brassicae, Lignum Millettiae, stephania tetrandra, radix liquiritiae. There is no study on elucidating the exact mechanism of Jiawei Yanghe decoction on OA so far. In present study, mechanisms of JWYHD effects on IL-1β-induced chondrocytes and in MIA-induced OA rats as anti-osteoarthritic were investigated. The present results showed that JWYHD increases protein and mRNA expressions of Col2a1 whereas decreases protein and mRNA

dependent manner and a concentration of 2.7 g/kg was most effective, whereas the concentration of 5.5 g/kg didn’t seem to ameliorate the cartilage degradation as shown in Fig.6B (P > 0.05). Considering the trend in western blot, ELISA and histopathological examination, JWYHD is viewed as an anti-osteoarthritic in vivo. Increasing its dosage beyond this, did not show more effect, suggesting that a potential optimal dose need to be identified. 4. Discussion In current conventional treatment of OA, non-steroid anti-inflammatory drugs possess no direct protection on the cartilage and is associated with adverse effects. Specific treatment of OA, such glucosamine, hyaluronic acid and platelet-rich plasma injection, are either controversial or not-recommended by guidelines due to their associated controversies. Therefore, in current situation, use of natural ways, including TCM formulas in treatment of OA has attracted much attention for its effectiveness on anti-inflammatory and absence of side effects [26]. Several classical TCM formulas in the treatment of OA, such as Bushen Huoxue decoction (BSHXD) and Duhuo Jisheng decoction (DHJSD) have been fully investigated their capacity on ameliorating OA progression and possible mechanisms determined [27–30]. Along with 7

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Fig. 4. Effects of JWYHD on protein and mRNA expressions of β-catenin, CyclinD1, Caspase-3, Caspase-9, MMP-3 and MMP-13 and Col2a1 in IL-1β-induced chondrocytes. Chondrocytes pre-treated with JWYHD at various concentrations (50, 100, 200, 400 μg/ml) followed by 10 ng/ml IL-1β stimulation for 12 h. (A) Protein blots of β-catenin, CyclinD1, Caspase-3, Caspase-9, MMP-3 and MMP-13 and Col2a1. (B) CyclinD1, (C) β-catenin, (D) Caspase-3, (E) Caspase-9, (F) MMP-3 and (G) MMP-13 were up-regulated in IL-1β-induced chondrocytes compared to the control. (H) Col2a1 was up-regulated in IL-1β-induced chondrocytes compared to the control. mRNA expressions of (I) CyclinD1, (J) β-catenin, (K) Caspase-3, (L) Caspase-9, (M) MMP-3 and (N) MMP-13 were up-regulated in IL-1β-induced chondrocytes compared to the control. (H) Col2a1 was up-regulated in IL-1β-induced chondrocytes compared to the control. JWYHD reversed these trends in a dosedependent manner. ##P < 0.01, compared to the control. *P < 0.05, **P < 0.01 compared to the IL-1β-induced chondrocytes. JWYHD reversed these observations in a dose-dependent manner. ##P < 0.01, compared to the control. *P < 0.05, **P < 0.01 compared to the IL-1β-induced chondrocytes.

changes. Cartilage degradation has essential role in the progression of OA and is characterized by chondrocyte apoptosis and extracellular matrix degradation [8,9]. Chondrocytes, the only cell type in the articular cartilage, regulate the homeostasis of cartilage with extracellular matrix of chondrocytes [7,9,37]. Besides, chondrocytes depend on the microenvironment provided by the extracellular matrix. Therefore, avoiding the chondrocytes apoptosis and extracellular matrix degradation might be a strategy for specific treatment of OA. It has been reported that OAlike inflammatory microenvironment results in chondrocytes apoptosis and excess production of MMPs, which results in the degradation of extracellular matrix [12,38]. IL-1β induction could reproduce similar inflammatory microenvironment like human OA. In this present study, IL-1β-induced OA-like changes on chondrocytes pathogenesis including chondrocytes apoptosis and extracellular matrix degradation. Our MTT assay results demonstrated that JWYHD improved the viability and proliferation of IL-1β-induced chondrocytes, which was further

expressions of Caspase-3, Caspase-9, MMP-3 and MMP-13 along with down-regulation of pro-inflammatory cytokines of IL-1β, IL-6 and TNFα via suppressing Wnt/β-catenin signaling pathway. This leads to antiapoptosis effect on chondrocytes and anti-degenerative effects on extracellular matrix. OA is characterized by degradation of articular cartilage, subchondral bone sclerosis, formation of osteophyte and synovitis [1]. Previous studies revealed that intra-articular injection of MIA into rat’s knee showed similar pathological effects as in human OA [24,31]. Therefore, replicating OA in rats is a well-accepted method identical to human OA [27,32–34]. IL-1β has a key role in the OA pathogenesis where it can enhance the expressions of MMPs and other metalloproteinase in chondrocytes. Meanwhile, IL-1β is the well-accepted identical to serve as induction for microenvironment of OA to establish a cellular OA model in vitro [35,36]. In this study, both MIA-induced rat model in vivo and IL-1β-induced cellular model significantly duplicated human OA like features and treatment with JWYHD reversed the induced 8

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Fig. 5. Effects of JWYHD on protein expressions and serum/synovium pro-inflammatory cytokines in MIA-induced OA rats. Rats received oral administration of various concentrations of JWYHD (1.4, 2.7 and 5.5 g/kg) for 8 weeks after intra-articular injection of MIA. Control group received saline injection and oral administration of saline. (A) Protein blots of (B) CyclinD1, (C) β-catenin, (D) Caspase-3, (E) Caspase-9, (F) MMP-3 and (G) MMP-13 and (H)Col2a1. The serum (A)IL-1β, (B)IL-6, (C)TNF-α and (D)synovium IL-1β, (E)IL-6 and (F)TNF-α were determined using ELISA following the manufacturer’s protocol. #P < 0.05, ##P < 0.01, ###P < 0.001 compared to the control. *P < 0.05, **P ＜ 0.01, ***P ＜ 0.001, compared to the MIA-induced OA rats.

effect was found under IL-1β-induced micro-inflammation condition. It has been found that not only progression of OA related with activation of Wnt signaling pathway, but also over suppression canonical Wnt signaling pathway also led to OA-like progression [21]. It is reasonable to assume that the mildly decrease on cell viability of JWYHD is caused by over suppression of Wnt signaling pathway. Under the inflammation condition, Wnt signaling pathway was activated and the Wnt suppression effect of JWYHD is benefitted to rebuild the homeostasis of

confirmed by DAPI staining assay. Alcian Blue staining illustrated that JWYHD maintained the integrity of extracellular matrix against IL-1βinduced inflammation. It was elucidated that the effects of JWYHD on the treatment of OA involve promotion of chondrocyte viability and preserving the integrity of extracellular matrix in an inflammatory microenvironment. In current study, interestingly, JWYHD showed no increase and even mildly decrease on cell viability, meanwhile significant increasing

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Fig. 6. JWYHD ameliorated cartilage degradation and arthritis index in MIA-induced OA rats. OA rats were induced by intraarticular injection of MIA (3 mg MIA in 50 μL) and then randomly designated to various concentration of JWYHD treated groups (1.4, 2.7, 5.5 g/kg) and received JWYHD by intragastric administration for 8 weeks. HematainEosion and Safranning O-fast green staining were conducted. (A) Histopathological observation, micro-CT and 3-Dimension remodeling and macroscopic observation was shown. (B) Modified Mankin scoring result was shown. (C) Percentage of the trabecular bone volume fraction (BV/TV). (D) #P＜0.05, compared to the control. *P ＜ 0.05, compared to the OA group. (D) Arthritis index of each group. #P < 0.05 compared to the control. *P < 0.05 compared to the MIA-induce OA rats.

progression of OA. Collagen Type Ⅱ is the most abundant and important collagen in the extracellular matrix of chondrocytes. MMP-3 cannot only break down collagen Type Ⅱ alone but are also capable of activating other MMPs like MMP-1 and MMP-13 [15]. MMP-13 is the most studied MMP and is shown to have the strongest ability in degrading collagen Type Ⅱ, it can cleave collagen Type Ⅱ 5 times more than MMP1 and 6 times more than MMP-3 [40,41]. MMPs can be induced by multiple ways including pro-inflammatory cytokines and Wnt/β-catenin signaling pathway [13,16,20,42]. Certain pro-inflammatory cytokines, such as IL-1β, IL-6, TNF-α, have been demonstrated to have fundamental roles in shifting physiological homeostasis of cartilage to pathological degradation of cartilage [43]. Caspase family, including

cartilage via suppressing activated Wnt signaling pathway. However, in normal condition, over dosage or duration of JWYHD treatment may suppress the regular metabolize of cartilage. In vivo study illustrated that the therapeutic effect didn’t increase with increasing dosage. In summary, the potential effect of JWYHD in treating OA may be limited to the pathological situation (like IL-1β stimulated micro inflammatory environment) and further study need to be done. MMPs, a family of proteases can degrade all components of extracellular matrix and plays an important role in OA progression [12,38,39]. MMP-3 and MMP-13, among MMPs family are the most prominent in degrading chondrocytes extracellular matrix. MMP-3 along with MMP-13 constitute the optimal microenvironment for the 10

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This study bears some insufficiencies due to certain limitations. First effects of JWYHD as anti-apoptosis on chondrocytes were observed through MTT assay and DAPI staining, instead of cell cycle detection, which impedes the consistency of the results. The microstructural observation was not performed due to lack of transmission electron microscopy observation. Furthermore, the optimal dosage of JWYHD in vivo is still not totally explored. Furthermore, a small interfering RNA is the best to investigate and validate the inhibitory effects on Wnt signaling pathway of JWYHD and is thus recommended.

Caspase-3 and Caspase-9, was found to be involved in multiple forms of chondrocyte apoptosis by initiating and executing cell death [44–46]. In the current study, JWYHD decreased the up-regulation of protein and mRNA expressions of β-catenin, CyclinD1, MMP-3, MMP-13, Caspase-3, Caspase-9 induced by IL-1β in a dose-dependent manner in vitro. Similar inhibitory effects were observed in JWYHD treated MIA-induced OA rats. Additionally, JWYHD decreased MIA-induced up-regulation of serum and synovium IL-1β, IL-6 and TNF-α in vivo. Surprisingly, effects of JWYHD did not increase with increasing dosage in vivo. Results demonstrated that JWYHD possess inhibitory effects on pro-apoptosis proteins, pro-degenerative metalloproteinases, and pro-inflammatory cytokines. Wnt/β-catenin signaling pathway is one of the multiples signaling pathways involved in pathogenesis of OA is important because of series of after effects of its aberrantly activation in pathogenesis of OA. Basically, Axin-1/2 and adenomatous polyposis coli (APC) assemble the steady degradation complex, consisting of casein kinase 1 (CK1), glycogen synthase kinase-3β (GSK-3β) and β-catenin. The degradation complex of β-catenin were normally degraded by the proteasome/ubiquitin-mediated pathway [47,48]. In the existence of Wnt ligands or Licl, a GSK-3β inhibitor, phosphorylation and degradation of β-catenin are suppressed and then the β-catenin accumulates in cytoplasm. Consequently, stabilized β-catenin translocate to the nucleus and activates transcription of the Wnt/β-catenin target genes including CyclinD1 [48]. Studies show that Wnt/β-catenin signaling pathway is involved in the catabolism of cartilage and hence development of OA [49,50]. Activation of Wnt/β-catenin signaling pathway stimulates abnormal gene and protein expression, including overexpression of MMPs and aggrecanases, as observed in OA models [17,51]. This leads to abnormal differentiation of chondrocytes [52] and degradation of extracellular matrix of cartilage [17], and hence the suppression of Wnt/β-catenin signaling pathway lowers the chondrocyte apoptosis and OA-like cartilage degradation [21,53]. These series of studies indicated that inhibition Wnt signaling pathway can be an option for the treatment of OA. However, over inhibition of Wnt/β-catenin signaling pathway also leads to OA progression [21]. Not much attention has been focused on the relationship between pro-inflammatory cytokines, pro-apoptosis related genes and Wnt/β-catenin signaling pathway. These relationships are investigated in this study. Licl was used as an activator of Wnt/β-catenin signaling pathway, consistent with previous studies, Wnt/β-catenin signaling pathway related protein expressions of β-catenin, CyclinD1, MMP-3 and MMP-13 was observed to be upregulated. Additionally, it was found that protein expressions of IL-1β, Caspase-3 and Caspase-9 were up-regulated on Licl-induced chondrocytes, which indicating that the possible mechanism of Wnt signaling pathway is not only by increasing MMPs but also by stimulating the chondrocytes apoptosis and up-regulation of pro-inflammatory cytokines. Moreover, not only inflammatory environment is able to stimulate Wnt/β-catenin signaling pathway but also the activation of Wnt signaling pathway leads to the up-regulation of IL-1β, which is in line with previous study. Yuasa et al. reported that Wnt/β-catenin signaling pathway produce MMPs and aggrecanases resulting in cartilage degradation and this had a potential up-regulating effect on activating IL1β [54]. DKK-1, a Wnt signaling pathway inhibitor, play a role in inhibition by competitive binding LPR-5/6 and was used in this study to be compare with JWYHD. Both JWYHD and DKK-1 inhibited up-regulation of β-catenin, CyclinD1, Caspase-3, Caspase-9, IL-1β, MMP-3 and MMP-13, but the inhibitory effect of JWYHD was more than DKK-1. It was inferred that the adverse effect of over inhibition on Wnt/β-catenin signaling pathway might be the reason why JWYHD could not show increasing effect with increasing dosage in vivo. On account of Wnt signaling pathway inhibition effect of JWYHD, excess dosage may lead to over inhibition of Wnt signaling pathway eventually resulting in OAlike progression. Results demonstrated that overdose of JWYHD in vivo is not beneficial to OA and an optimal dose needs to be further determined.

5. Conclusion In conclusion, JWYHD promoted chondrocyte viability against apoptosis, decreased MMP-3, MMP-13, Caspase-3, Caspase-9 via Wnt/βcatenin signaling pathway in both IL-1β-induced and Licl-induced chondrocytes. Similar inhibitory effects were reproduced in the MIAinduced OA rat model, along with the downregulation of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. Additionally, histopathological observations validated the amelioration of JWYHD treated MIA-induced OA rats. Altogether, JWYHD possess anti-osteoarthritic and chondroprotective effect in the treatment of OA in vitro and in vivo. Availability of data and materials No additionally unpublished data were used to support this study. Declaration of Competing Interest The authors declare that they have no competing interests. Acknowledgement This study was supported by the Health commission of Jiangxi Province (TCM famous master Xu HZ studio), the Traditional Chinese Medicine foundation of Health commission of Jiangxi Province (2018A371) and the Postgraduate Innovation Foundation of Jiangxi Province (YC2017-S363). Special funds for the Construction of firstclass disciplines in Jiangxi Province (JXSYLXK-ZHYI012). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.109708. References [1] W.K. Aicher, B. Rolauffs, The spatial organisation of joint surface chondrocytes: review of its potential roles in tissue functioning, disease and early, preclinical diagnosis of osteoarthritis, Ann. Rheum. Dis. 73 (4) (2014) 645-53. [2] X. Tang, S. Wang, S. Zhan, et al., The Prevalence of Symptomatic Knee Osteoarthritis in China: Results From the China Health and Retirement Longitudinal Study, Null 68 (3) (2016) 648-53. [3] J.W.J. Bijlsma, F. Berenbaum, F.P.J.G. Lafeber, Osteoarthritis: An update with relevance for clinical practice, Lancet 377 (9783) (2011) 2115–2126. [4] Q. Liu, J. Niu, J. Huang, et al., Knee osteoarthritis and all-cause mortality: the Wuchuan Osteoarthritis Study, Osteoarthr. Cartil. 23 (7) (2015) 1154-7. [5] Q. Liu, J. Niu, H. Li, et al., Knee Symptomatic Osteoarthritis, Walking Disability, NSAIDs Use and All-cause Mortality: Population-based Wuchuan Osteoarthritis Study, Sci. Rep. 7 (1) (2017). [6] R.B. Sim, G.A. Hawker, R. Croxford, et al., All-Cause Mortality and Serious Cardiovascular Events in People with Hip and Knee Osteoarthritis: A Population Based Cohort Study, PLoS One 9 (3) (2014) e91286. [7] F. Guilak, R. Nims, A. Dicks, et al., Osteoarthritis as a disease of the cartilage pericellular matrix[J], Matrix Biol. 71–72 (2018) 40–50. [8] M.B. Goldring, The role of the chondrocyte in osteoarthritis, Arthritis Rheum. 43 (9) (2000) 1916-26. [9] A. Reddi, Aging, osteoarthritis and transforming growth factor-beta signaling in cartilage, Arthritis Res. Ther. 8 (1) (2006) 101. [10] D. Mistry, Y. Oue, M.G. Chambers, M.V. Kayser, et al., Chondrocyte death during murine osteoarthritis, Osteoarthr. Cartil. 12 (2) (2004) 131-41. [11] R. Loeser, Aging and osteoarthritis: the role of chondrocyte senescence and aging

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