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Chondroprotective and antiarthritic effects of Daphnetin used in vitro and in vivo osteoarthritis models
Journal Pre-proof Chondroprotective and antiarthritic effects of daphnetin used in vitro and in vivo osteoarthritis models
Xiaohan Zhang, Jun Yao, Zhengyuan Wu, Kai Zou, Zhenyi Yang, Xing Huang, Zhiwei Luan, Jia Li, Qingjun Wei PII:
S0024-3205(19)30784-2
DOI:
https://doi.org/10.1016/j.lfs.2019.116857
Reference:
LFS 116857
To appear in:
Life Sciences
Received date:
4 May 2019
Revised date:
6 September 2019
Accepted date:
6 September 2019
Please cite this article as: X. Zhang, J. Yao, Z. Wu, et al., Chondroprotective and antiarthritic effects of daphnetin used in vitro and in vivo osteoarthritis models, Life Sciences(2019), https://doi.org/10.1016/j.lfs.2019.116857
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© 2019 Published by Elsevier.
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Chondroprotective and Antiarthritic Effects of Daphnetin u s e d In Vitro and In Vivo Osteoarthritis Models Xiaohan Zhang1#, Jun Yao2,3#, Zhengyuan Wu1, Kai Zou1, Zhenyi Yang3, Xing Huang3, Zhiwei Luan3, Jia Li4*, Qingjun Wei1,2* 1
Department of Orthopedics Trauma and Hand Surgery, Guangxi Medical University First Affiliated
Hospital, Guangxi Medical University, Nanning, China; 2
Guangxi Collaborative Innovation Center for Biomedicine, Guangxi Medical University, Nanning,
China 3
Department of Bone and Joint Surgery, Guangxi Medical University First Affiliated Hospital,
Guangxi Medical University, Nanning, China; Department of Pathology, Guangxi Medical University First Affiliated Hospital, Guangxi Medical
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University, Nanning, China; #Contributed equally
*Corresponding Authors: Qingjun Wei and Jia Li: The First Affiliated Hospital of Guangxi Medical
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University, Guangxi Medical University, Nanning, 530021, China; E-mail: [email protected] (Q.W.), [email protected] (J.L.); Tel.: +86-07715350189; Fax: +86-07715350189. Present address:
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Shuangyong Road No.22, Nanning Guangxi, China.
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Abstract Aims Daphnetin (DAP) is a traditional Chinese drug usually used to treat cardiovascular diseases. Studies have confirmed the anti-inflammatory, antioxidant, anti-bacterial and insecticidal, anti-tumor and neuro-protective effects of DAP. However, its anti-arthritic potential remains unexplored. The aim of this study is to investigate the in vitro and in vivo chondroprotective effects of DAP. Main methods The effect of DAP on primary rabbit chondrocytes was examined using recombinant human IL-1β for 24 hours. For the in vivo studies, rabbits were randomly divided into groups: a normal control group and osteoarthritis (OA) groups. The OA groups received three different doses of DAP for 4 or 8 weeks. The anti-arthritic effect of DAP was assessed using histopathological examinations, qRT-PCR, western blotting and immunohistochemical analysis. Key findings Both in vitro and in vivo results indicate that DAP exerts a protective effect against IL-1β in chondrocytes. In vitro, DAP inhibits the expression of IL-6, IL-12, MMP-3, MMP-9 and MMP-13, induced by IL-1β in rabbit chondrocytes, and stimulates the production of IL-10. The inhibitory effect of DAP on the MMPs is partially regulated by the inhibition of the PI3K/AKT, MAPK and NF-κB signaling pathways. The effect of DAP on OA may be attributed to the suppression of inflammatory factor secretion, chondrocyte apoptosis observed by the decrease in pro-apoptotic Caspase-3 and BAX, and the activation of anti-apoptotic BCL-2. Significance 1 / 15
Journal Pre-proof DAP has a broad range of prospects in the treatment of OA, which provides a novel therapeutic strategy for OA. Keywords Daphnetin; chondroprotective; anti-arthritic; chondrocytes; osteoarthritis
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1 Introduction Osteoarthritis (OA) is a degenerative disease of the joints that is characterized by pain and limited function; radiographic manifestations of osteophyte formation and joint space narrowing; and pathological changes in cartilage and subchondral bone integrity [1]. The prevalence in the population over 65 years old is about 60% in males, and about 70% in females [2]. Moreover, the incidence of OA is on the rise due to an aging population, lifestyle choices, dietary habits, obesity, malnutrition, occupational injury and trauma. The activation of injury-induced catabolic factors contributes to the progression of OA [3, 4]. Chondrocyte exposure to IL-1β and matrix metalloproteinases (MMPs) released by TNF-α also facilitate cartilage degeneration [5]. Accumulating evidence indicates that MMPs play important roles in cartilage degeneration given their ability to degrade extracellular matrix (ECM) [6]. Various pathways, including MAPK [7], NF-κB [8], and PI3K/Akt signaling also facilitate the expression of MMPs during the course of OA. Drugs currently used for OA, such as analgesics, opioids, and NSAIDs [9], merely relieve the symptoms of pain and inflammation, fail to prevent disease progression and are associated with several side effects, including gastrointestinal bleeding [10]. Recently, plant derivatives with minimal side effects and cost-effectiveness have received considerable attention and are becoming a new hotspot in the study of OA adjuvant therapy [11, 12] . Daphnetin (DAP), 7, 8-dihydroxycoumarin [13], is a traditional Chinese drug, which is usually used for the treatment of cardiovascular diseases. A large number of studies have confirmed that DAP has anti-inflammatory [14], anti-bacterial, insecticidal [15], anti-carcinogenic [16] and neuroprotective properties [17]. Although the active ingredients and general safety in the use of DAP have been well recognized, its anti-arthritic properties remain unexplored. The aim of this study is to determine the in vitro and in vivo effectiveness of DAP in mitigating inflammation, ameliorating cartilage damage and articular pathology. 2 Materials & Methods 2.1 Materials Daphnetin (DAP, CAS: 486-35-1) was purchased from Chengdu Must Biological Technology Co. Ltd. and was dissolved in sterile phosphate-buffered saline (PBS) to obtain a 100 mg/ml solution and stored at -20℃, in a refrigerator. Interleukin (IL)-1β was dissolved in sterile PBS to obtain a 0.1 mg/ml solution and was also stored at -20℃. RNA primers were dissolved in sterile PBS, following the instructions given, and stored at 4℃. All primary antibodies were purchased from Abcam PLC and were diluted in chilled PBS at a ratio of 1:1,000 or 1:50 and stored at -20℃. 2.2 Establishment of Rabbit Osteoarthritis (OA)Models 2 / 15
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Sixty New Zealand white rabbits (3-month-old, male, 2.0±0.2 kg) were purchased from the Animal Center of Guangxi Medical University, Nanning, China. All experimental procedures were approved by the Animal Ethics Committee of Guangxi Medical University (Certificate No. 201805008). The rabbits were randomly split into 2 experimental groups: a normal control group (n=12) and an OA group (n=48). Eight weeks after surgery, the rabbits in the OA group were randomly divided into 4 groups: positive control (n=12) and therapy groups (n=36). The therapy groups received an intra-articular 0.2 mL injection of 12, 24 or 48 ng/mL DAP, for 4 or 8 weeks. The osteoarthritis model was created following the method reported by Rogart et al. [18]. All rabbits were intramuscularly injected with 400,000 units of penicillin for three consecutive days post-surgery, daily. All procedures were performed by a single surgeon in a blinded fashion. The rabbits were kept in single-subject cages. One rabbit died on the 4th day after surgery, and two unoperated rabbits died on the 8th and the 17th day, respectively. Three rabbits were then added and randomly modeled to meet the group requirements. 2.3 Sample Collection After anesthesia (as described before) [18], 15 mL of blood was collected from the femoral artery of each rabbit at 4 and 8 weeks after the drug knee injection therapy, and kept for 60 minutes at 4℃, and the serum was collected by centrifuging the blood samples at 1,000 ×g for 15 minutes at 4℃, and the sample was stored at -80℃ until further use. The anesthetized animals were sacrificed by injecting air through the ear vein, and knee joints were harvested and either fixed in 4% paraformaldehyde for histopathological examination or stored at -80°C for downstream analyses. 2.4 Histopathological and Immunohistochemistry (IHC) The fixed tissues were decalcified and paraffin embedded for histological and IHC analyses. 5 μm sections were stained with Hematoxylin-Eosin (HE), Masson’s trichrome, Safranin O and were microscopically evaluated for histopathological changes using a modified scoring system, as described in Colombo et al. [19]. The specific scoring standards used are shown in Table 1. Immunohistochemical staining was performed based on instructions given on the kit (Solarbio, Beijing, China), and was observed under a microscope (Olympus, Tokyo, Japan). 2.5 Sandwich Enzyme-linked Immunosorbent Assay (ELISA) for Serum Cytokines Circulating MMP-13, IL-6, and TNF-α in rabbit serum were measured using sandwich ELISA kits (CUSABIO BIOTECH CO., LTD, Wuhan, China), according to the manufacturer’s instructions. 2.6 Isolation and Culture of primary Chondrocytes This animal study was approved by the Animal Ethics Committee of Guangxi Medical University (Certificate No.:201805009). For isolation of primary chondrocytes, rabbits (n=2) were picked out within 72 hours of birth, sacrificed through cervical dislocation, immersed in 75% alcohol, and the limbs were removed on a clean bench and then immersed in PBS containing 10% penicillin–streptomycin. Muscle tissues and cartilages from the hip, knee and ankle joints were dissected out and placed on a petri dish containing PBS with 10% penicillin–streptomycin, cut into 3 / 15
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1mm³ pieces, and washed three times with PBS. The tissues were initially digested with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) for 30 minutes, followed by 0.2% Collagenase-2 (Solarbio, Beijing, China) in Dulbecco’s modified Eagle’s medium (DMEM) for 4 hours at 37℃. The digested tissue was centrifuged at 400 ×g for 5 minutes, and after discarding the supernatant, articular chondrocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 50 U/mL penicillin, and 50 μg/mL streptomycin, and cultured in 5% CO2 at 37℃. The chondrocytes were grown to 90% confluency and passaged not more than 3 times. For all in vitro manipulations, the chondrocytes were detached using 3 mL trypsin-EDTA and were pelleted through centrifugation (100 ×g for 5 min). 2.7 Cell seeding Cells at passage 3 and at about 70%-80% confluency were trypsinized and pelleted through centrifugation. The cells were seeded in 24-well plates (5,000 cells per well) and 6-well plates (20,000 cells per well), and cultured for 24 hours. In order to facilitate staining, the cells were planted in a 24-well plate with a circular cell climber. 2.8 Cytotoxicity Measurement and Treatment Procedures Chondrocytes were collected at log phase and plated in 96-well plates at 5,000 cells/200 μL per well. An equal amount of PBS was added into the control wells. The plates were incubated at 37℃ and 5% CO2 in an incubator. The cells with 60% confluency were treated with different concentrations of DAP (3 replicates per group) and allowed to grow for 24 hours and cytotoxicity was assessed using an MTT assay (Solarbio, Beijing, China), following the manufacturer’s instructions. Absorbance was measured at 490 nm on a multi-function microplate reader. We selected three appropriate concentrations of co-culture with IL-1β (10 mL/kg) for 24 hours to assess the markers of osteoarthritis. IL-1β is a known mediator of inflammation and cartilage degradation [20]. We used the following equation to evaluate the proliferation effect of DAP: Proliferation effect [%] = 100 x (the number of living cells treated with DAP at various concentrations)/ (the number of living cells treated without DAP). 2.9 Total Ribonucleic Acid (RNA) Isolation Total RNA from cultured chondrocytes and articular cartilages were extracted using a Total RNA Extraction Kit, following the manufacturer’s instructions (Aidlab, Beijing, China). Before RNA extraction, articular cartilages were ground using a mortar and a small amount of liquid nitrogen. RNA concentration was measured on a Nanodrop 2000 reader. 2.10 Total protein extraction Total protein from cells and tissues was extracted, following the manufacturer’s instructions on the total protein extraction kit. Cells were trypsinized in a medium in which the culture dish was aspirated, digested with trypsin and transferred into a 1.5 mL centrifuge tube, and 350 μL of lysate and 3.5 μL of phenylmethylsulfonyl fluoride (PMSF) were added. 3.5 μL of a protease inhibitor phosphatase inhibitor mixture was added, after the sample was digested in a refrigerator at 4℃ for half an hour. The supernatant was aspirated through centrifugation at 15,000 ×g for 25 minutes in a 4℃ centrifuge and the absorbed supernatant was stored at -80℃. Protein concentration 4 / 15
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was measured using a BCA test kit (Solarbio, Beijing, China). 2.11 Gene Expression The effect of DAP on the osteoarthritis-associated expression of MMPs and interleukins in chondrocytes was investigated using quantitative Real-Time PCR (qRT-PCR). 2.1 μg of RNA was reverse transcribed into cDNA using a PrimeScript RT reagent Kit gDNA Eraser (TAKARA BIO INC. Kusatsu, Shiga, Japan). Each 20 μL reaction mixture contained 3.5 μL of template cDNA, 10 μL of PowerUpTM SYBR® GreenMaster Mix (Appliedbiosystem™, Thermo Fisher Scientific, Vilnius, Lithuania), and 200 nM of primers. qRT-PCR was performed on specifically amplified products that were determined using melting curve analysis. Primers used to detect gene expression are listed in Table 2. 2.12 Western Blotting Analysis Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis buffers were used to boil the sample for 10 minutes at 100 degrees Celsius, and 60 μL of each sample was separated on 10% polyacrylamide gel and transferred onto a PVDF membrane. After blocking with 5% bovine serum albumin (BSA), the samples were incubated with primary antibodies targeting P38, p-P38, ERK, p-ERK, Akt, p-Akt, PI3K, p-PI3K, IκB, NF-κB P65, p-P65 and β-Actin, overnight at 4℃. After extensive washing with 0.1% TBST, the membranes were incubated with secondary antibodies (1:10,000) for 1 hour at room temperature. After the immunoblotting reaction, protein bands were detected using chemiluminescence (ECL) reagents and observed using an image analyzer (LiuorChemTM FC3, protein simple, San Jose, CA, USA). Equal volumes of the ECL (Pierce™ ECL Western Blotting Substrate, Thermo Scientific™, Rockford, IL, USA) chemiluminescence reagent A solution and B solution were mixed together. After 1 min, the mixture was added to the surface of the blotting membrane and incubated for 5 min. Absorbent paper was used to remove residual luminescence reagents, and they were wrapped in plastic film to avoid air bubbles forming, and the contact surface of the blotting film and the film were kept dry. Once the film was placed on a blotting film, the film position was fixed, and the exposure clip was quickly closed. Under red light, the exposure time was adjusted according to the intensity of the fluorescence. The film was taken out and rapidly developed through immersion, and development was terminated after a band appeared. Image analysis was conducted using ImageJ software, after the images were captured using AlphaView software (LiuorChemTM FC3, protein simple, San Jose, CA, USA). By measuring the gray value of images, the relative expression levels of the phosphorylated proteins (p-P38, p-ERK, p-Akt, p-PI3K and p-P65) and total form proteins (P38, ERK, Akt, PI3K, NF-κB P65, IκB and β-Actin) were quantitatively obtained and compared with that of the control. 2.13 Chondrocyte Morphology Cell slides were removed from 24-well plates, washed 3 times with PBS for 3 minutes each, fixed in 4% paraformaldehyde for 20 minutes, washed with PBS another 3 times for 3 minutes each, and stained with Hematoxylin-Eosin (HE), Saffron O and toluidine blue. Changes in chondrocyte morphology, including cytoskeletal actin fibers (red), and nuclear aggregations (blue) of the chondrocytes 5 / 15
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were observed under a microscope (Olympus, Tokyo, Japan). Changes in red, shallow, and qualitative cytoskeletal actin fibers stained by Safranin O were observed. In the toluidine blue staining, the nuclear aggregation rate was qualitatively determined using the nuclear blue staining interval. 2.14 Immunofluorescence Cell slides were taken out of the 24-well plate, washed 3 times with PBS for 3 minutes each, fixed in 4% paraformaldehyde for 20 minutes, washed with PBS another 3 times for 3 minutes each. Staining was performed following the manufacturer’s instructions on the immunofluorescence kit (Beyotime, Wuhan, China), and observed under a blue light laser microscope. When combined with TNF-α antibodies and secondary antibodies with specific markers, the cytoplasm with these indicators were found to be positive for red fluorescence and negative for no red fluorescence. 2.15 Detection of chondrocyte apoptosis Chondrocyte apoptosis was measured using TUNEL, following the manufacturer’s instructions. Apoptotic cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan) using a standard fluorescein filter to observe the green fluorescence of fluorescein at 520 ± 20 nm. 2.16 Statistical Analysis Data are expressed as mean ± standard deviation (SD). One-way ANOVA with Bonferroni post-test was used to assess the differences among groups using SPSS 24.0 to analyze these differences. Statistical significance was set at p<0.05.
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3 Results 3.1 Cell Viability The effect of DAP on chondrocyte proliferation was determined using MTT. All experiments were performed in triplicate. Different doses of DAP (5-50 ng/mL) were screened to select its optimal concentration for the growth of chondrocytes. Based on the preliminary screening, we chose 12, 24 and 48 ng/ml DAP for further experiments (Fig. 1A). 3.2 The Effect of DAP on Chondrocytes TUNEL assay was performed to elucidate the effect of the three doses of DAP on chondrocyte apoptosis (Fig. 1B). Since TUNEL assay is only a semi-quantitative test with relatively large errors, we also performed quantitative measurements at genetic level. The expression of apoptosis-related genes were determined using qRT-PCR (Fig. 1C-G). In chondrocytes treated with IL-1β and in cartilage tissue of the OA groups, DAP was found to have decreased the expression of Caspase-3 and BAX, while BCL-2 expression was upregulated. Therefore, it was found that DAP decreases the expression of pro-apoptotic Caspase-3 and BAX and increase the expression level of anti-apoptotic BCL-2. Out of the three concentrations tested, 12 ng/ml for 8 weeks yielded the optimal treatment effect. 3.3 Effect of DAP on Chondrocyte Morphology Morphological changes in chondrocytes were observed using HE and Safranin O, toluidine blue staining (Fig. 2A). HE staining demonstrated the typical polygonal 6 / 15
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morphology of chondrocytes when cultured in a single layer. Relatively more cell colonies were found in the DAP group compared with the IL-1β group or the control groups. The cytoskeletal actin fibers (red) and the cell aggregated nuclei (blue) of the chondrocytes were observed using Safranin O and toluidine blue staining, respectively. We found that DAP-treated groups had denser cells than the IL-1β group. Cytoskeletal actin fibers and denser cells with aggregated nuclei are consistent with the results of HE staining. Among them, the 12 ng/ml group showed the highest level of aggregation. 3.4 The Effect of DAP on the Expression of Cartilage-specific Markers. qRT-PCR was performed to study the effects of different doses of DAP on the expression of chondrocytes markers and chondrocyte apoptosis markers. In vitro, DAP was found to have reversed the IL-1β induced decrease in the expression of ACAN, SOX9 and Collagen-2 (Fig. 2B). However, Collagen-1 was found to be downregulated by DAP, suggesting that DAP may delay or prevent the dedifferentiation of chondrocytes in vitro. In vivo, 8 weeks of treatment with 12 ng/ml DAP elicited the highest expression of ACAN/SOX9/Collagen-2 and the lowest expression of Collagen-1 (Fig. 2C-F). 3.5 Effect of DAP on Articular Cartilage We constructed a surgically induced OA experimental animal model to assess the effect of DAP on OA pathology. In the normal group, the cartilage on the femoral condyle was found to have a smooth surface without any observable defects or osteophyte formation (Fig. 3A). In the OA group, characteristic features of OA, such as erosion and callus formation, were observed in the femoral condyle. Progressive destruction of cartilage was also observed. Contemporaneously, DAP therapy reduced cartilage damage seen in the OA model group, and prolonged treatment with DAP mitigated cartilage wear. Overall, DAP treatment was found to decrease the macroscopic score at week 4 and 8, compared with the OA model group, in a time-dependent manner (Fig. 3D-G). 3.6 Histopathological examination Histopathological examination using HE showed that compared with the normal control group, higher infiltration of mononuclear cells, increased fibrosis and an irregular cartilage surface and defects (Fig. 3B-C), chondrocyte degeneration, and evidence of cloning and focal hypercytosis, were observed in the OA group (Fig. 3B-C). Masson staining and safranin-green staining showed further fissures, ulcers, defects, proteoglycans and synovial tissue hyperplasia in the surface layer, which was significantly greater in the OA group than the control group. These pathological changes were found to have been decreased through DAP therapy (Fig. 3B-C). In addition, 8 weeks of 12 ng/mL DAP treatment resulted in better cartilage protection than any other dose, as assessed by the presence of fissures, ulcers (or erosion), and synovial hyperplasia (Fig. 3D-G). 3.7 Effects of DAP on inflammatory and anti-inflammatory factors in OA Both in vivo and in vitro, transcript abundance of inflammatory factors IL-6, IL-12, MMP-3, MMP-9 and MMP-13 were significantly downregulated and anti-inflammatory IL-10 was upregulated in rabbits treated with DAP, compared with 7 / 15
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rabbits of the OA model group (p< 0.05; Fig. 4A-4H). Immunohistochemical staining of MMP-1 and TNF-α showed significantly different results between the normal, model and therapy groups (Fig. 4I-J). The effect of DAP on serum MMP-13, IL-6 and TNF-α were determined by quantifying their expression levels in serum before and after treatment, in the model and therapy groups, as shown in Fig. 4K-M. qRT-PCR, IHC and ELISA demonstrated a similar result. In the treatment groups, inflammatory factors IL-6, IL-12, MMP-3, MMP-9 and MMP-13 were observed to be significantly downregulated, with rabbits receiving 12 ng/mL exhibiting the strongest effect (P<0.05). Hence, the 24 and 48 ng/mL treatment groups were found to be less effective. However, significant differences were still observed in these treatment groups compared with that of the serum of rabbits in the disease model group. Longer the therapy period the higher the number of differences. 3.8 Immunofluorescence DAPI staining showed intact nuclei and uniform chromatin. Disintegrated nuclei with aggregation around the periphery was evident by the intense and granular red fluorescence in IL-1β-treated cells (Fig. 4N), compared with the uniform but weak fluorescence in the normal group, indicating that normal chondrocytes have a low level of TNF-α expression. Inconsistent intensity of fluorescence observed in different DAP treatment groups indicate the differential expression of TNF-α among these groups. 3.9 Signaling Pathway In vitro studies show that DAP decreased the IL-1β-induced increase in p-P38/P38, p-ERK/ERK, p-PI3K/PI3K, p-P65/P65 and IκB levels (Fig. 5A). DAP also reverses the IL-1β induced reduction of p-Akt/Akt levels. At a concentration of 12 ng/ml, DAP elicits its maximal effect on rabbits of the treatment groups (Fig. 5B). 3.10 Side-effects of DAP During the entire experiment, three rabbits died, and necropsy was performed to assess the cause of death in consultation with the Animal Experiment Center. The death of the model animals may be related to the local climate and had nothing to do with the experiment. In addition, we did not observe any side effects or adverse reactions to DAP in the animal models. 4 Discussion In OA, inflammation is accompanied by connective tissue hyperplasia, destruction of articular cartilage and subchondral bone [21]. Therefore, treatment with inflammatory factors can relieve pain and swelling [20]. Drug therapy is the only available option for treating OA [22]. DAP, as an activator of blood and a stasis removal agent, has been used to treat cardiovascular diseases [23, 24], However, its effects on inflammatory chemokines and cytokine expression has remained unexplored. MMPs play a key role in cartilage degradation by regulating the degradation of several extracellular matrix components, such as aggregator, fibronectin and MMP-13, and especially Collagen-2 [25]. In vitro, DAP inhibits the expression of IL-6, IL-12, MMP-3, MMP-9 and MMP-13 induced by IL-1β in rabbit chondrocytes at gene level and stimulates the production of IL-10. This result indicates that the cartilage protective effect of DAP may be related to the regulation of 8 / 15
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MMPs. Similarly, in vivo studies have shown that DAP decreases the circulating levels of TNF-α, IL-6 and MMP-13. In this study, the expression of both ACAN and Collagen-2 was found to have improved in DAP-treated chondrocytes, probably due to its anti-inflammatory and MMPs-regulating effects. As our study shows, chondrogenesis is induced when Medial Collateral Ligament (MCL) and Anterior Cruciate Ligament (ACL) are cut-off, due to mechanical instability [26]. ECM components (such as Collagen-2, ACAN and MMPs) play important roles in maintaining the structural, as well as functional integrity, of the joints. Our results show that treatment with DAP for a period can ameliorate the structure of the damaged joint and increase the expression of cartilage-specific markers. Specifically, the mRNA expression of TNF-α, IL-6 and MMP-1, MMP-3, MMP-9 and MMP-13 were found to have decreased, while IL-10 expression was found to have increased in the OA model animals treated with DAP, and this change was both time and dose dependent (Fig. 2-4). These results are consistent with that of in vitro studies (Fig. 4), suggesting that DAP is effective both in vitro and in vivo. The anti- arthritic potential of DAP is related to several signaling pathways. NF-κB is at the center of inflammation and accelerates the progression of ECM injury and cartilage destruction [27]. In the pathophysiological process of OA, inflammatory factors activate the NF-κB dimer through phosphorylation of its inhibitor, IkB. IκB is an inhibitory protein of NF-κB [28], which is phosphorylated and degraded by plastids. This enables NF-κB to enter the caryon in order to accelerate the transcription of inflammatory genes, including TNF-α [29]. However, DAP significantly downregulates the expression of NF-κB and IκB (Fig. 5A-B). It is well known that P38 is a kinase that regulates the activity of NF-κB. P38 plays a key role in the intracellular signal transduction pathway activated by pro-inflammatory factors associated with the etiology of OA [30]. Based on our research, DAP can effectively reduce various inflammatory factors, due to the expression levels of various inflammatory factors. As a result of the reduced expression of various inflammatory factors, it inhibits the expression of IκB and the phosphorylation activation of NF-κB and P38. As a result, the NF-κB signaling pathway is inhibited and the anti-inflammatory effect of DAP is enhanced [31-33]. Activation of downstream MAPK pathway can specifically upregulate the expression of TNF-α and MMP-13 [27, 34, 35], supporting the role of DAP for the regulation of the MAPK pathway. The PI3K/AKT signaling pathway also plays a role in OA (Fig. 5A-5B). The chondroprotective effect has been revealed through histopathological examination. The therapeutic effect of DAP is, however, not dose dependent. 12 ng/ml DAP for 8 weeks was found to be more effective than 48 ng/ml for 4 weeks, possibly due to cytotoxicity at the higher concentration. 5 Conclusions In conclusion, DAP was found to show a protective effect on chondrocytes treated with IL-1β in vitro, and animal models of OA in vivo. The anti-arthritic effect of DAP can be attributed to the inhibition of PI3K/AKT, MAPK and NF-κB signaling pathway and the reduction of apoptotic Caspase-3 and BAX, parallel to the activation 9 / 15
Journal Pre-proof of anti-apoptotic BCL-2. These results suggest that DAP has a broad range of prospects for the treatment of OA and provides a novel therapeutic intervention for OA. However, further research is needed to gain better insight into its mechanism.
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Abbreviations DAP: Daphnetin OA: Osteoarthritis RNA: Ribonucleic Acid ECM: Extracellular Matrix MAPK: Mitogen-Activated Protein Kinase EDTA: Trypsin-Ethylenediaminetetraacetic Acid PMSF: Phenylmethylsulfonyl Fluoride ELISA: Sandwich Enzyme-linked Immunosorbent Assay qRT-PCR: Quantitative Real-Time quantitative polymerase chain reaction WB: Western Bolt DAPI: 4',6-diamidino-2-phenylindole TUNEL: Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling PVDF: Polyvinylidene Fluoride BCL-2: B-Cell Lymphoma-2 Caspase-3: Cysteinyl Aspartate Specific Proteinase TNF: Tumor Necrosis Factor IL: Interleukin MMP: Matrix Metalloprotease COL: Collagen ERK: Signal-regulated kinase AKT: Protein Kinase B PI3K: Phosphatidylinositol-3 Kinase NF-κB: Nuclear Factor Kappa B IκB: Inhibitor of Nuclear Factor Kappa B β-Actin: beta actin GADPH: Glyceraldehyde-3-phosphate dehydrogenase MCL: Medial Collateral Ligament ACL: Anterior Cruciate Ligament
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Availability of Data & Materials All data supporting our findings are contained within the manuscript.
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Acknowledgments: This study was supported by the National Natural Science Foundation of China, the Key Program of Guangxi Collaborative Innovation Center for Biomedicine, the Guangxi Natural Science Foundation, the Guangxi Scientific Research and Technology Development Program, the Guangxi Health, Family Planning Commission Program, the Guangxi Key R&D Program and Self-financing research project of Guangxi Zhuang Region Health Department. Funding The National Natural Science Foundation of China (81760390, 81760402, and 81560371) The Key Program of Guangxi Collaborative Innovation Center for Biomedicine (GCICB‐SR‐2017005) The Guangxi Natural Science Foundation (2017GXNSFAA198258, 2015GXNSFBA139158 and 2018GXNSFAA294075) The Guangxi Scientific Research and Technology Development Program (S201655 and S201668) 10 / 15
Journal Pre-proof The Guangxi Health and Family Planning Commission Program (GZLC‐16–32) The Guangxi Key R&D Program (GuiKeAB17292073) Self-financing research project of Guangxi Zhuang Region Health Department (Z2016742 and Z20180941) Notes #Xiaohan Zhang and Jun Yao contributed equally to this work.
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Author’s Contributions Xiaohan Zhang, Qingjun Wei, Jun Yao, Jia Li and Zhengyuan Wu conceived and designed the study; Xiaohan Zhang, Zhengyuan Wu, Kai Zou, Zhenyi Yang, Xing Huang and Zhiwei Luan collected and analyzed the data; Xiaohan Zhang and Zhengyuan Wu contributed reagents/materials/analysis tools; and Xiaohan Zhang and Qingjun Wei wrote the paper.
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Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. References
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35.
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Minimal
Mild
Moderate
Marked
Loss of superficial layer
0
1
2
3
4
Ulcer or erosion
0
1
2
3
4
Fissure
0
1
2
3
4
Loss of proteoglycan
0
1
2
3
4
Synovial tissue hyperplasia
0
1
2
3
4
Table 1. Histopathological scoring system
Forward
Reverse
TGA GTA CCT GAA CCG 3t
GAT GAC GCT GGA CTT CCT CCG AGA G 3t 5t CTA AGC CAC GGT GAT GAA GGA GTC 3t 5t GAG GCA CTG GCG GAA GTC AAT C 3t 5t GCC AAG CCT TGT CGG AGA TGA TC 3t 5t GCA CTC GAC CGC TCA GCA TG 3t 5t GTT CCT TGG CTT GGA GGT GAC AG 3t 5t GTG AAG ACG CAG ACG GTG GAT TC 3t 5t TCT ACA CCT ACA CCG GCA AGA GTC3t 5t GAG GCA CTG GCG GAA GTC AAT C 3t 5tTCA AGT GCT CCA GCA AGA ACA AG 3t 5t GAA GAA CTG GTG GA CAG CAA GAG 3t 5t GCG TAG CCT ACA TGG ACC AAC AG 3t 5t AGA CAC GAT GGT GAA GGT CG 3t
5t CCG TAC AGT TCC ACG AAG
5t GAC
GCA TC 3t
5t AGA TGG TGA GTG AGG CGG TGA G 3t 5t CAC TGT CTG TCT CGA TGC CAC TG 3t CTC AGC AGG CAG GTC 3t 5tCTG CTC CAC TGC CTT GCT CTT G 3t 5tAAG CCA GGC AAC TCT CAT TCT TGG 3t 5tCAA T G G CAG CAT CAA CAG CAT CAT C 3t 5tGGT ACT CAC ACG CCA GAA GAA GC 3t 5t CGG AGA CTG GTA ATG GCA TCA AGG 3t 5tGAA GTG ATT CTC AGC AGG CAG GTC 3t 5tCTC CGC CTC CTC CAC GAA GG 3t 5tTGA AGT GGA AGC CGC CAT TGA TG 3t 5t AGC CGT CGT GGA GGA CAG TG 3t 5t TGC CGT GGG TGG AAT CAT AC 3t
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5tGAA GTG ATT
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Table 2. Primer sequences utilized for qRT-PCR evaluation of gene expression.
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BCL-2 BAX caspase-3 IL-6 IL-10 IL-12 MMP-3 MMP-9 MMP-13 ACAN SOX-9 COL-2 COL-1 GAPDH
5t GTG
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Journal Pre-proof Figure 1: Chondro-protective effects of DAP on IL-1β- induced chondrocytes. (A) MTT assay was to the effect of DAP on cartilage proliferation (A). (Control: without any treatment; *p < 0.05, **p < 0.01, ***p < 0.001). (B) TUNEL measured chondrocyte apoptosis. qRT-PCR was performed to determine the expression levels of (C) BCL-2, BAX, BCL-2/BAX and Caspase-3 in vitro and (D-G) in vivo. Bars with different letters are significantly different from each other at P < 0.05. Bars with different letters are significantly different from each other at P < 0.05 (original low magnification ×200 (scale bar, 400 μm)). Figure 2. Effect of DAP on the expression of specific markers. Chondrocytes were pretreated with DAP followed by IL-1β (10 ng/ mL). qRT-PCR was performed to determine the expression levels of (A) Hematoxylin-eosin and toluidine blue and safranin O stained morphology. (B) ACAN, SOX-9, Collagen-2, Collagen-1 in vitro and (C-F) in vivo.
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Figure 3. Effect of DAP on the treatment of OA. (A) Macroscopic appearance. (B) Hematoxylin-eosin staining, Masson staining, and safranin-green staining were performed in sections of cartilage. (C-F) Histological score of articular cartilage was determined. Bars with different letters are significantly different from each other at P < 0.05 (original magnification × 200 (scale bar, 400 μm)).
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Figure 4. Effect of DAP on the treatment of inflammatory factor in OA. (A-H) qRT-PCR was used to analyze the expression of IL-6, IL-10, IL-12, MMP-3, MMP-9 and MMP-13 genes in vitro and vivo. Bars with different letters are significantly different from each other at P < 0.05 . (I-J) Immunohistochemical staining of MMP-1 and TNF-α was performed in sections of cartilage (original magnification × 200 (scale bar, 400 μm) ). (K-M) ELISA measured inflammatory factorrelative factors (MMP-13, IL-6 and TNF-α) from serum. Bars with different letters are significantly different from each other at P < 0.05. (N) Immunofluorescence of TNF-α was performed in sections of chondrocytes (original magnification × 200 (scale bar, 400 μm) ).
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Figure 5. Signaling pathway research. (A) Western Blot was used to analyze the progression of the expression of PI3K/AKT, MAPK, and NF-κB signaling pathway proteins P38, p-P38, ERK, p-ERK, Akt, p-Akt, PI3K, p-PI3K, NF-κB P65, p-P65 and IκB (the gels have been run under the same experimental conditions). (B) Schematic description of relative signaling pathways that are activated by DAP. Bars with different letters are significantly different from each other at P < 0.05.
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Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Xiaohan Zhang, Jun Yao, Zhengyuan Wu, Kai Zou, Zhenyi Yang, Xing Huang, Zhiwei Luan, Jia Li, Qingjun Wei PII:
S0024-3205(19)30784-2
DOI:
https://doi.org/10.1016/j.lfs.2019.116857
Reference:
LFS 116857
To appear in:
Life Sciences
Received date:
4 May 2019
Revised date:
6 September 2019
Accepted date:
6 September 2019
Please cite this article as: X. Zhang, J. Yao, Z. Wu, et al., Chondroprotective and antiarthritic effects of daphnetin used in vitro and in vivo osteoarthritis models, Life Sciences(2019), https://doi.org/10.1016/j.lfs.2019.116857
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Chondroprotective and Antiarthritic Effects of Daphnetin u s e d In Vitro and In Vivo Osteoarthritis Models Xiaohan Zhang1#, Jun Yao2,3#, Zhengyuan Wu1, Kai Zou1, Zhenyi Yang3, Xing Huang3, Zhiwei Luan3, Jia Li4*, Qingjun Wei1,2* 1
Department of Orthopedics Trauma and Hand Surgery, Guangxi Medical University First Affiliated
Hospital, Guangxi Medical University, Nanning, China; 2
Guangxi Collaborative Innovation Center for Biomedicine, Guangxi Medical University, Nanning,
China 3
Department of Bone and Joint Surgery, Guangxi Medical University First Affiliated Hospital,
Guangxi Medical University, Nanning, China; Department of Pathology, Guangxi Medical University First Affiliated Hospital, Guangxi Medical
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4
University, Nanning, China; #Contributed equally
*Corresponding Authors: Qingjun Wei and Jia Li: The First Affiliated Hospital of Guangxi Medical
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University, Guangxi Medical University, Nanning, 530021, China; E-mail: [email protected] (Q.W.), [email protected] (J.L.); Tel.: +86-07715350189; Fax: +86-07715350189. Present address:
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Shuangyong Road No.22, Nanning Guangxi, China.
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Abstract Aims Daphnetin (DAP) is a traditional Chinese drug usually used to treat cardiovascular diseases. Studies have confirmed the anti-inflammatory, antioxidant, anti-bacterial and insecticidal, anti-tumor and neuro-protective effects of DAP. However, its anti-arthritic potential remains unexplored. The aim of this study is to investigate the in vitro and in vivo chondroprotective effects of DAP. Main methods The effect of DAP on primary rabbit chondrocytes was examined using recombinant human IL-1β for 24 hours. For the in vivo studies, rabbits were randomly divided into groups: a normal control group and osteoarthritis (OA) groups. The OA groups received three different doses of DAP for 4 or 8 weeks. The anti-arthritic effect of DAP was assessed using histopathological examinations, qRT-PCR, western blotting and immunohistochemical analysis. Key findings Both in vitro and in vivo results indicate that DAP exerts a protective effect against IL-1β in chondrocytes. In vitro, DAP inhibits the expression of IL-6, IL-12, MMP-3, MMP-9 and MMP-13, induced by IL-1β in rabbit chondrocytes, and stimulates the production of IL-10. The inhibitory effect of DAP on the MMPs is partially regulated by the inhibition of the PI3K/AKT, MAPK and NF-κB signaling pathways. The effect of DAP on OA may be attributed to the suppression of inflammatory factor secretion, chondrocyte apoptosis observed by the decrease in pro-apoptotic Caspase-3 and BAX, and the activation of anti-apoptotic BCL-2. Significance 1 / 15
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1 Introduction Osteoarthritis (OA) is a degenerative disease of the joints that is characterized by pain and limited function; radiographic manifestations of osteophyte formation and joint space narrowing; and pathological changes in cartilage and subchondral bone integrity [1]. The prevalence in the population over 65 years old is about 60% in males, and about 70% in females [2]. Moreover, the incidence of OA is on the rise due to an aging population, lifestyle choices, dietary habits, obesity, malnutrition, occupational injury and trauma. The activation of injury-induced catabolic factors contributes to the progression of OA [3, 4]. Chondrocyte exposure to IL-1β and matrix metalloproteinases (MMPs) released by TNF-α also facilitate cartilage degeneration [5]. Accumulating evidence indicates that MMPs play important roles in cartilage degeneration given their ability to degrade extracellular matrix (ECM) [6]. Various pathways, including MAPK [7], NF-κB [8], and PI3K/Akt signaling also facilitate the expression of MMPs during the course of OA. Drugs currently used for OA, such as analgesics, opioids, and NSAIDs [9], merely relieve the symptoms of pain and inflammation, fail to prevent disease progression and are associated with several side effects, including gastrointestinal bleeding [10]. Recently, plant derivatives with minimal side effects and cost-effectiveness have received considerable attention and are becoming a new hotspot in the study of OA adjuvant therapy [11, 12] . Daphnetin (DAP), 7, 8-dihydroxycoumarin [13], is a traditional Chinese drug, which is usually used for the treatment of cardiovascular diseases. A large number of studies have confirmed that DAP has anti-inflammatory [14], anti-bacterial, insecticidal [15], anti-carcinogenic [16] and neuroprotective properties [17]. Although the active ingredients and general safety in the use of DAP have been well recognized, its anti-arthritic properties remain unexplored. The aim of this study is to determine the in vitro and in vivo effectiveness of DAP in mitigating inflammation, ameliorating cartilage damage and articular pathology. 2 Materials & Methods 2.1 Materials Daphnetin (DAP, CAS: 486-35-1) was purchased from Chengdu Must Biological Technology Co. Ltd. and was dissolved in sterile phosphate-buffered saline (PBS) to obtain a 100 mg/ml solution and stored at -20℃, in a refrigerator. Interleukin (IL)-1β was dissolved in sterile PBS to obtain a 0.1 mg/ml solution and was also stored at -20℃. RNA primers were dissolved in sterile PBS, following the instructions given, and stored at 4℃. All primary antibodies were purchased from Abcam PLC and were diluted in chilled PBS at a ratio of 1:1,000 or 1:50 and stored at -20℃. 2.2 Establishment of Rabbit Osteoarthritis (OA)Models 2 / 15
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Sixty New Zealand white rabbits (3-month-old, male, 2.0±0.2 kg) were purchased from the Animal Center of Guangxi Medical University, Nanning, China. All experimental procedures were approved by the Animal Ethics Committee of Guangxi Medical University (Certificate No. 201805008). The rabbits were randomly split into 2 experimental groups: a normal control group (n=12) and an OA group (n=48). Eight weeks after surgery, the rabbits in the OA group were randomly divided into 4 groups: positive control (n=12) and therapy groups (n=36). The therapy groups received an intra-articular 0.2 mL injection of 12, 24 or 48 ng/mL DAP, for 4 or 8 weeks. The osteoarthritis model was created following the method reported by Rogart et al. [18]. All rabbits were intramuscularly injected with 400,000 units of penicillin for three consecutive days post-surgery, daily. All procedures were performed by a single surgeon in a blinded fashion. The rabbits were kept in single-subject cages. One rabbit died on the 4th day after surgery, and two unoperated rabbits died on the 8th and the 17th day, respectively. Three rabbits were then added and randomly modeled to meet the group requirements. 2.3 Sample Collection After anesthesia (as described before) [18], 15 mL of blood was collected from the femoral artery of each rabbit at 4 and 8 weeks after the drug knee injection therapy, and kept for 60 minutes at 4℃, and the serum was collected by centrifuging the blood samples at 1,000 ×g for 15 minutes at 4℃, and the sample was stored at -80℃ until further use. The anesthetized animals were sacrificed by injecting air through the ear vein, and knee joints were harvested and either fixed in 4% paraformaldehyde for histopathological examination or stored at -80°C for downstream analyses. 2.4 Histopathological and Immunohistochemistry (IHC) The fixed tissues were decalcified and paraffin embedded for histological and IHC analyses. 5 μm sections were stained with Hematoxylin-Eosin (HE), Masson’s trichrome, Safranin O and were microscopically evaluated for histopathological changes using a modified scoring system, as described in Colombo et al. [19]. The specific scoring standards used are shown in Table 1. Immunohistochemical staining was performed based on instructions given on the kit (Solarbio, Beijing, China), and was observed under a microscope (Olympus, Tokyo, Japan). 2.5 Sandwich Enzyme-linked Immunosorbent Assay (ELISA) for Serum Cytokines Circulating MMP-13, IL-6, and TNF-α in rabbit serum were measured using sandwich ELISA kits (CUSABIO BIOTECH CO., LTD, Wuhan, China), according to the manufacturer’s instructions. 2.6 Isolation and Culture of primary Chondrocytes This animal study was approved by the Animal Ethics Committee of Guangxi Medical University (Certificate No.:201805009). For isolation of primary chondrocytes, rabbits (n=2) were picked out within 72 hours of birth, sacrificed through cervical dislocation, immersed in 75% alcohol, and the limbs were removed on a clean bench and then immersed in PBS containing 10% penicillin–streptomycin. Muscle tissues and cartilages from the hip, knee and ankle joints were dissected out and placed on a petri dish containing PBS with 10% penicillin–streptomycin, cut into 3 / 15
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1mm³ pieces, and washed three times with PBS. The tissues were initially digested with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) for 30 minutes, followed by 0.2% Collagenase-2 (Solarbio, Beijing, China) in Dulbecco’s modified Eagle’s medium (DMEM) for 4 hours at 37℃. The digested tissue was centrifuged at 400 ×g for 5 minutes, and after discarding the supernatant, articular chondrocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 50 U/mL penicillin, and 50 μg/mL streptomycin, and cultured in 5% CO2 at 37℃. The chondrocytes were grown to 90% confluency and passaged not more than 3 times. For all in vitro manipulations, the chondrocytes were detached using 3 mL trypsin-EDTA and were pelleted through centrifugation (100 ×g for 5 min). 2.7 Cell seeding Cells at passage 3 and at about 70%-80% confluency were trypsinized and pelleted through centrifugation. The cells were seeded in 24-well plates (5,000 cells per well) and 6-well plates (20,000 cells per well), and cultured for 24 hours. In order to facilitate staining, the cells were planted in a 24-well plate with a circular cell climber. 2.8 Cytotoxicity Measurement and Treatment Procedures Chondrocytes were collected at log phase and plated in 96-well plates at 5,000 cells/200 μL per well. An equal amount of PBS was added into the control wells. The plates were incubated at 37℃ and 5% CO2 in an incubator. The cells with 60% confluency were treated with different concentrations of DAP (3 replicates per group) and allowed to grow for 24 hours and cytotoxicity was assessed using an MTT assay (Solarbio, Beijing, China), following the manufacturer’s instructions. Absorbance was measured at 490 nm on a multi-function microplate reader. We selected three appropriate concentrations of co-culture with IL-1β (10 mL/kg) for 24 hours to assess the markers of osteoarthritis. IL-1β is a known mediator of inflammation and cartilage degradation [20]. We used the following equation to evaluate the proliferation effect of DAP: Proliferation effect [%] = 100 x (the number of living cells treated with DAP at various concentrations)/ (the number of living cells treated without DAP). 2.9 Total Ribonucleic Acid (RNA) Isolation Total RNA from cultured chondrocytes and articular cartilages were extracted using a Total RNA Extraction Kit, following the manufacturer’s instructions (Aidlab, Beijing, China). Before RNA extraction, articular cartilages were ground using a mortar and a small amount of liquid nitrogen. RNA concentration was measured on a Nanodrop 2000 reader. 2.10 Total protein extraction Total protein from cells and tissues was extracted, following the manufacturer’s instructions on the total protein extraction kit. Cells were trypsinized in a medium in which the culture dish was aspirated, digested with trypsin and transferred into a 1.5 mL centrifuge tube, and 350 μL of lysate and 3.5 μL of phenylmethylsulfonyl fluoride (PMSF) were added. 3.5 μL of a protease inhibitor phosphatase inhibitor mixture was added, after the sample was digested in a refrigerator at 4℃ for half an hour. The supernatant was aspirated through centrifugation at 15,000 ×g for 25 minutes in a 4℃ centrifuge and the absorbed supernatant was stored at -80℃. Protein concentration 4 / 15
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was measured using a BCA test kit (Solarbio, Beijing, China). 2.11 Gene Expression The effect of DAP on the osteoarthritis-associated expression of MMPs and interleukins in chondrocytes was investigated using quantitative Real-Time PCR (qRT-PCR). 2.1 μg of RNA was reverse transcribed into cDNA using a PrimeScript RT reagent Kit gDNA Eraser (TAKARA BIO INC. Kusatsu, Shiga, Japan). Each 20 μL reaction mixture contained 3.5 μL of template cDNA, 10 μL of PowerUpTM SYBR® GreenMaster Mix (Appliedbiosystem™, Thermo Fisher Scientific, Vilnius, Lithuania), and 200 nM of primers. qRT-PCR was performed on specifically amplified products that were determined using melting curve analysis. Primers used to detect gene expression are listed in Table 2. 2.12 Western Blotting Analysis Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis buffers were used to boil the sample for 10 minutes at 100 degrees Celsius, and 60 μL of each sample was separated on 10% polyacrylamide gel and transferred onto a PVDF membrane. After blocking with 5% bovine serum albumin (BSA), the samples were incubated with primary antibodies targeting P38, p-P38, ERK, p-ERK, Akt, p-Akt, PI3K, p-PI3K, IκB, NF-κB P65, p-P65 and β-Actin, overnight at 4℃. After extensive washing with 0.1% TBST, the membranes were incubated with secondary antibodies (1:10,000) for 1 hour at room temperature. After the immunoblotting reaction, protein bands were detected using chemiluminescence (ECL) reagents and observed using an image analyzer (LiuorChemTM FC3, protein simple, San Jose, CA, USA). Equal volumes of the ECL (Pierce™ ECL Western Blotting Substrate, Thermo Scientific™, Rockford, IL, USA) chemiluminescence reagent A solution and B solution were mixed together. After 1 min, the mixture was added to the surface of the blotting membrane and incubated for 5 min. Absorbent paper was used to remove residual luminescence reagents, and they were wrapped in plastic film to avoid air bubbles forming, and the contact surface of the blotting film and the film were kept dry. Once the film was placed on a blotting film, the film position was fixed, and the exposure clip was quickly closed. Under red light, the exposure time was adjusted according to the intensity of the fluorescence. The film was taken out and rapidly developed through immersion, and development was terminated after a band appeared. Image analysis was conducted using ImageJ software, after the images were captured using AlphaView software (LiuorChemTM FC3, protein simple, San Jose, CA, USA). By measuring the gray value of images, the relative expression levels of the phosphorylated proteins (p-P38, p-ERK, p-Akt, p-PI3K and p-P65) and total form proteins (P38, ERK, Akt, PI3K, NF-κB P65, IκB and β-Actin) were quantitatively obtained and compared with that of the control. 2.13 Chondrocyte Morphology Cell slides were removed from 24-well plates, washed 3 times with PBS for 3 minutes each, fixed in 4% paraformaldehyde for 20 minutes, washed with PBS another 3 times for 3 minutes each, and stained with Hematoxylin-Eosin (HE), Saffron O and toluidine blue. Changes in chondrocyte morphology, including cytoskeletal actin fibers (red), and nuclear aggregations (blue) of the chondrocytes 5 / 15
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were observed under a microscope (Olympus, Tokyo, Japan). Changes in red, shallow, and qualitative cytoskeletal actin fibers stained by Safranin O were observed. In the toluidine blue staining, the nuclear aggregation rate was qualitatively determined using the nuclear blue staining interval. 2.14 Immunofluorescence Cell slides were taken out of the 24-well plate, washed 3 times with PBS for 3 minutes each, fixed in 4% paraformaldehyde for 20 minutes, washed with PBS another 3 times for 3 minutes each. Staining was performed following the manufacturer’s instructions on the immunofluorescence kit (Beyotime, Wuhan, China), and observed under a blue light laser microscope. When combined with TNF-α antibodies and secondary antibodies with specific markers, the cytoplasm with these indicators were found to be positive for red fluorescence and negative for no red fluorescence. 2.15 Detection of chondrocyte apoptosis Chondrocyte apoptosis was measured using TUNEL, following the manufacturer’s instructions. Apoptotic cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan) using a standard fluorescein filter to observe the green fluorescence of fluorescein at 520 ± 20 nm. 2.16 Statistical Analysis Data are expressed as mean ± standard deviation (SD). One-way ANOVA with Bonferroni post-test was used to assess the differences among groups using SPSS 24.0 to analyze these differences. Statistical significance was set at p<0.05.
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3 Results 3.1 Cell Viability The effect of DAP on chondrocyte proliferation was determined using MTT. All experiments were performed in triplicate. Different doses of DAP (5-50 ng/mL) were screened to select its optimal concentration for the growth of chondrocytes. Based on the preliminary screening, we chose 12, 24 and 48 ng/ml DAP for further experiments (Fig. 1A). 3.2 The Effect of DAP on Chondrocytes TUNEL assay was performed to elucidate the effect of the three doses of DAP on chondrocyte apoptosis (Fig. 1B). Since TUNEL assay is only a semi-quantitative test with relatively large errors, we also performed quantitative measurements at genetic level. The expression of apoptosis-related genes were determined using qRT-PCR (Fig. 1C-G). In chondrocytes treated with IL-1β and in cartilage tissue of the OA groups, DAP was found to have decreased the expression of Caspase-3 and BAX, while BCL-2 expression was upregulated. Therefore, it was found that DAP decreases the expression of pro-apoptotic Caspase-3 and BAX and increase the expression level of anti-apoptotic BCL-2. Out of the three concentrations tested, 12 ng/ml for 8 weeks yielded the optimal treatment effect. 3.3 Effect of DAP on Chondrocyte Morphology Morphological changes in chondrocytes were observed using HE and Safranin O, toluidine blue staining (Fig. 2A). HE staining demonstrated the typical polygonal 6 / 15
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morphology of chondrocytes when cultured in a single layer. Relatively more cell colonies were found in the DAP group compared with the IL-1β group or the control groups. The cytoskeletal actin fibers (red) and the cell aggregated nuclei (blue) of the chondrocytes were observed using Safranin O and toluidine blue staining, respectively. We found that DAP-treated groups had denser cells than the IL-1β group. Cytoskeletal actin fibers and denser cells with aggregated nuclei are consistent with the results of HE staining. Among them, the 12 ng/ml group showed the highest level of aggregation. 3.4 The Effect of DAP on the Expression of Cartilage-specific Markers. qRT-PCR was performed to study the effects of different doses of DAP on the expression of chondrocytes markers and chondrocyte apoptosis markers. In vitro, DAP was found to have reversed the IL-1β induced decrease in the expression of ACAN, SOX9 and Collagen-2 (Fig. 2B). However, Collagen-1 was found to be downregulated by DAP, suggesting that DAP may delay or prevent the dedifferentiation of chondrocytes in vitro. In vivo, 8 weeks of treatment with 12 ng/ml DAP elicited the highest expression of ACAN/SOX9/Collagen-2 and the lowest expression of Collagen-1 (Fig. 2C-F). 3.5 Effect of DAP on Articular Cartilage We constructed a surgically induced OA experimental animal model to assess the effect of DAP on OA pathology. In the normal group, the cartilage on the femoral condyle was found to have a smooth surface without any observable defects or osteophyte formation (Fig. 3A). In the OA group, characteristic features of OA, such as erosion and callus formation, were observed in the femoral condyle. Progressive destruction of cartilage was also observed. Contemporaneously, DAP therapy reduced cartilage damage seen in the OA model group, and prolonged treatment with DAP mitigated cartilage wear. Overall, DAP treatment was found to decrease the macroscopic score at week 4 and 8, compared with the OA model group, in a time-dependent manner (Fig. 3D-G). 3.6 Histopathological examination Histopathological examination using HE showed that compared with the normal control group, higher infiltration of mononuclear cells, increased fibrosis and an irregular cartilage surface and defects (Fig. 3B-C), chondrocyte degeneration, and evidence of cloning and focal hypercytosis, were observed in the OA group (Fig. 3B-C). Masson staining and safranin-green staining showed further fissures, ulcers, defects, proteoglycans and synovial tissue hyperplasia in the surface layer, which was significantly greater in the OA group than the control group. These pathological changes were found to have been decreased through DAP therapy (Fig. 3B-C). In addition, 8 weeks of 12 ng/mL DAP treatment resulted in better cartilage protection than any other dose, as assessed by the presence of fissures, ulcers (or erosion), and synovial hyperplasia (Fig. 3D-G). 3.7 Effects of DAP on inflammatory and anti-inflammatory factors in OA Both in vivo and in vitro, transcript abundance of inflammatory factors IL-6, IL-12, MMP-3, MMP-9 and MMP-13 were significantly downregulated and anti-inflammatory IL-10 was upregulated in rabbits treated with DAP, compared with 7 / 15
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rabbits of the OA model group (p< 0.05; Fig. 4A-4H). Immunohistochemical staining of MMP-1 and TNF-α showed significantly different results between the normal, model and therapy groups (Fig. 4I-J). The effect of DAP on serum MMP-13, IL-6 and TNF-α were determined by quantifying their expression levels in serum before and after treatment, in the model and therapy groups, as shown in Fig. 4K-M. qRT-PCR, IHC and ELISA demonstrated a similar result. In the treatment groups, inflammatory factors IL-6, IL-12, MMP-3, MMP-9 and MMP-13 were observed to be significantly downregulated, with rabbits receiving 12 ng/mL exhibiting the strongest effect (P<0.05). Hence, the 24 and 48 ng/mL treatment groups were found to be less effective. However, significant differences were still observed in these treatment groups compared with that of the serum of rabbits in the disease model group. Longer the therapy period the higher the number of differences. 3.8 Immunofluorescence DAPI staining showed intact nuclei and uniform chromatin. Disintegrated nuclei with aggregation around the periphery was evident by the intense and granular red fluorescence in IL-1β-treated cells (Fig. 4N), compared with the uniform but weak fluorescence in the normal group, indicating that normal chondrocytes have a low level of TNF-α expression. Inconsistent intensity of fluorescence observed in different DAP treatment groups indicate the differential expression of TNF-α among these groups. 3.9 Signaling Pathway In vitro studies show that DAP decreased the IL-1β-induced increase in p-P38/P38, p-ERK/ERK, p-PI3K/PI3K, p-P65/P65 and IκB levels (Fig. 5A). DAP also reverses the IL-1β induced reduction of p-Akt/Akt levels. At a concentration of 12 ng/ml, DAP elicits its maximal effect on rabbits of the treatment groups (Fig. 5B). 3.10 Side-effects of DAP During the entire experiment, three rabbits died, and necropsy was performed to assess the cause of death in consultation with the Animal Experiment Center. The death of the model animals may be related to the local climate and had nothing to do with the experiment. In addition, we did not observe any side effects or adverse reactions to DAP in the animal models. 4 Discussion In OA, inflammation is accompanied by connective tissue hyperplasia, destruction of articular cartilage and subchondral bone [21]. Therefore, treatment with inflammatory factors can relieve pain and swelling [20]. Drug therapy is the only available option for treating OA [22]. DAP, as an activator of blood and a stasis removal agent, has been used to treat cardiovascular diseases [23, 24], However, its effects on inflammatory chemokines and cytokine expression has remained unexplored. MMPs play a key role in cartilage degradation by regulating the degradation of several extracellular matrix components, such as aggregator, fibronectin and MMP-13, and especially Collagen-2 [25]. In vitro, DAP inhibits the expression of IL-6, IL-12, MMP-3, MMP-9 and MMP-13 induced by IL-1β in rabbit chondrocytes at gene level and stimulates the production of IL-10. This result indicates that the cartilage protective effect of DAP may be related to the regulation of 8 / 15
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MMPs. Similarly, in vivo studies have shown that DAP decreases the circulating levels of TNF-α, IL-6 and MMP-13. In this study, the expression of both ACAN and Collagen-2 was found to have improved in DAP-treated chondrocytes, probably due to its anti-inflammatory and MMPs-regulating effects. As our study shows, chondrogenesis is induced when Medial Collateral Ligament (MCL) and Anterior Cruciate Ligament (ACL) are cut-off, due to mechanical instability [26]. ECM components (such as Collagen-2, ACAN and MMPs) play important roles in maintaining the structural, as well as functional integrity, of the joints. Our results show that treatment with DAP for a period can ameliorate the structure of the damaged joint and increase the expression of cartilage-specific markers. Specifically, the mRNA expression of TNF-α, IL-6 and MMP-1, MMP-3, MMP-9 and MMP-13 were found to have decreased, while IL-10 expression was found to have increased in the OA model animals treated with DAP, and this change was both time and dose dependent (Fig. 2-4). These results are consistent with that of in vitro studies (Fig. 4), suggesting that DAP is effective both in vitro and in vivo. The anti- arthritic potential of DAP is related to several signaling pathways. NF-κB is at the center of inflammation and accelerates the progression of ECM injury and cartilage destruction [27]. In the pathophysiological process of OA, inflammatory factors activate the NF-κB dimer through phosphorylation of its inhibitor, IkB. IκB is an inhibitory protein of NF-κB [28], which is phosphorylated and degraded by plastids. This enables NF-κB to enter the caryon in order to accelerate the transcription of inflammatory genes, including TNF-α [29]. However, DAP significantly downregulates the expression of NF-κB and IκB (Fig. 5A-B). It is well known that P38 is a kinase that regulates the activity of NF-κB. P38 plays a key role in the intracellular signal transduction pathway activated by pro-inflammatory factors associated with the etiology of OA [30]. Based on our research, DAP can effectively reduce various inflammatory factors, due to the expression levels of various inflammatory factors. As a result of the reduced expression of various inflammatory factors, it inhibits the expression of IκB and the phosphorylation activation of NF-κB and P38. As a result, the NF-κB signaling pathway is inhibited and the anti-inflammatory effect of DAP is enhanced [31-33]. Activation of downstream MAPK pathway can specifically upregulate the expression of TNF-α and MMP-13 [27, 34, 35], supporting the role of DAP for the regulation of the MAPK pathway. The PI3K/AKT signaling pathway also plays a role in OA (Fig. 5A-5B). The chondroprotective effect has been revealed through histopathological examination. The therapeutic effect of DAP is, however, not dose dependent. 12 ng/ml DAP for 8 weeks was found to be more effective than 48 ng/ml for 4 weeks, possibly due to cytotoxicity at the higher concentration. 5 Conclusions In conclusion, DAP was found to show a protective effect on chondrocytes treated with IL-1β in vitro, and animal models of OA in vivo. The anti-arthritic effect of DAP can be attributed to the inhibition of PI3K/AKT, MAPK and NF-κB signaling pathway and the reduction of apoptotic Caspase-3 and BAX, parallel to the activation 9 / 15
Journal Pre-proof of anti-apoptotic BCL-2. These results suggest that DAP has a broad range of prospects for the treatment of OA and provides a novel therapeutic intervention for OA. However, further research is needed to gain better insight into its mechanism.
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Abbreviations DAP: Daphnetin OA: Osteoarthritis RNA: Ribonucleic Acid ECM: Extracellular Matrix MAPK: Mitogen-Activated Protein Kinase EDTA: Trypsin-Ethylenediaminetetraacetic Acid PMSF: Phenylmethylsulfonyl Fluoride ELISA: Sandwich Enzyme-linked Immunosorbent Assay qRT-PCR: Quantitative Real-Time quantitative polymerase chain reaction WB: Western Bolt DAPI: 4',6-diamidino-2-phenylindole TUNEL: Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling PVDF: Polyvinylidene Fluoride BCL-2: B-Cell Lymphoma-2 Caspase-3: Cysteinyl Aspartate Specific Proteinase TNF: Tumor Necrosis Factor IL: Interleukin MMP: Matrix Metalloprotease COL: Collagen ERK: Signal-regulated kinase AKT: Protein Kinase B PI3K: Phosphatidylinositol-3 Kinase NF-κB: Nuclear Factor Kappa B IκB: Inhibitor of Nuclear Factor Kappa B β-Actin: beta actin GADPH: Glyceraldehyde-3-phosphate dehydrogenase MCL: Medial Collateral Ligament ACL: Anterior Cruciate Ligament
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Availability of Data & Materials All data supporting our findings are contained within the manuscript.
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Acknowledgments: This study was supported by the National Natural Science Foundation of China, the Key Program of Guangxi Collaborative Innovation Center for Biomedicine, the Guangxi Natural Science Foundation, the Guangxi Scientific Research and Technology Development Program, the Guangxi Health, Family Planning Commission Program, the Guangxi Key R&D Program and Self-financing research project of Guangxi Zhuang Region Health Department. Funding The National Natural Science Foundation of China (81760390, 81760402, and 81560371) The Key Program of Guangxi Collaborative Innovation Center for Biomedicine (GCICB‐SR‐2017005) The Guangxi Natural Science Foundation (2017GXNSFAA198258, 2015GXNSFBA139158 and 2018GXNSFAA294075) The Guangxi Scientific Research and Technology Development Program (S201655 and S201668) 10 / 15
Journal Pre-proof The Guangxi Health and Family Planning Commission Program (GZLC‐16–32) The Guangxi Key R&D Program (GuiKeAB17292073) Self-financing research project of Guangxi Zhuang Region Health Department (Z2016742 and Z20180941) Notes #Xiaohan Zhang and Jun Yao contributed equally to this work.
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Author’s Contributions Xiaohan Zhang, Qingjun Wei, Jun Yao, Jia Li and Zhengyuan Wu conceived and designed the study; Xiaohan Zhang, Zhengyuan Wu, Kai Zou, Zhenyi Yang, Xing Huang and Zhiwei Luan collected and analyzed the data; Xiaohan Zhang and Zhengyuan Wu contributed reagents/materials/analysis tools; and Xiaohan Zhang and Qingjun Wei wrote the paper.
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Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. References
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35.
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Minimal
Mild
Moderate
Marked
Loss of superficial layer
0
1
2
3
4
Ulcer or erosion
0
1
2
3
4
Fissure
0
1
2
3
4
Loss of proteoglycan
0
1
2
3
4
Synovial tissue hyperplasia
0
1
2
3
4
Table 1. Histopathological scoring system
Forward
Reverse
TGA GTA CCT GAA CCG 3t
GAT GAC GCT GGA CTT CCT CCG AGA G 3t 5t CTA AGC CAC GGT GAT GAA GGA GTC 3t 5t GAG GCA CTG GCG GAA GTC AAT C 3t 5t GCC AAG CCT TGT CGG AGA TGA TC 3t 5t GCA CTC GAC CGC TCA GCA TG 3t 5t GTT CCT TGG CTT GGA GGT GAC AG 3t 5t GTG AAG ACG CAG ACG GTG GAT TC 3t 5t TCT ACA CCT ACA CCG GCA AGA GTC3t 5t GAG GCA CTG GCG GAA GTC AAT C 3t 5tTCA AGT GCT CCA GCA AGA ACA AG 3t 5t GAA GAA CTG GTG GA CAG CAA GAG 3t 5t GCG TAG CCT ACA TGG ACC AAC AG 3t 5t AGA CAC GAT GGT GAA GGT CG 3t
5t CCG TAC AGT TCC ACG AAG
5t GAC
GCA TC 3t
5t AGA TGG TGA GTG AGG CGG TGA G 3t 5t CAC TGT CTG TCT CGA TGC CAC TG 3t CTC AGC AGG CAG GTC 3t 5tCTG CTC CAC TGC CTT GCT CTT G 3t 5tAAG CCA GGC AAC TCT CAT TCT TGG 3t 5tCAA T G G CAG CAT CAA CAG CAT CAT C 3t 5tGGT ACT CAC ACG CCA GAA GAA GC 3t 5t CGG AGA CTG GTA ATG GCA TCA AGG 3t 5tGAA GTG ATT CTC AGC AGG CAG GTC 3t 5tCTC CGC CTC CTC CAC GAA GG 3t 5tTGA AGT GGA AGC CGC CAT TGA TG 3t 5t AGC CGT CGT GGA GGA CAG TG 3t 5t TGC CGT GGG TGG AAT CAT AC 3t
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5tGAA GTG ATT
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Table 2. Primer sequences utilized for qRT-PCR evaluation of gene expression.
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BCL-2 BAX caspase-3 IL-6 IL-10 IL-12 MMP-3 MMP-9 MMP-13 ACAN SOX-9 COL-2 COL-1 GAPDH
5t GTG
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Journal Pre-proof Figure 1: Chondro-protective effects of DAP on IL-1β- induced chondrocytes. (A) MTT assay was to the effect of DAP on cartilage proliferation (A). (Control: without any treatment; *p < 0.05, **p < 0.01, ***p < 0.001). (B) TUNEL measured chondrocyte apoptosis. qRT-PCR was performed to determine the expression levels of (C) BCL-2, BAX, BCL-2/BAX and Caspase-3 in vitro and (D-G) in vivo. Bars with different letters are significantly different from each other at P < 0.05. Bars with different letters are significantly different from each other at P < 0.05 (original low magnification ×200 (scale bar, 400 μm)). Figure 2. Effect of DAP on the expression of specific markers. Chondrocytes were pretreated with DAP followed by IL-1β (10 ng/ mL). qRT-PCR was performed to determine the expression levels of (A) Hematoxylin-eosin and toluidine blue and safranin O stained morphology. (B) ACAN, SOX-9, Collagen-2, Collagen-1 in vitro and (C-F) in vivo.
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Figure 3. Effect of DAP on the treatment of OA. (A) Macroscopic appearance. (B) Hematoxylin-eosin staining, Masson staining, and safranin-green staining were performed in sections of cartilage. (C-F) Histological score of articular cartilage was determined. Bars with different letters are significantly different from each other at P < 0.05 (original magnification × 200 (scale bar, 400 μm)).
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Figure 4. Effect of DAP on the treatment of inflammatory factor in OA. (A-H) qRT-PCR was used to analyze the expression of IL-6, IL-10, IL-12, MMP-3, MMP-9 and MMP-13 genes in vitro and vivo. Bars with different letters are significantly different from each other at P < 0.05 . (I-J) Immunohistochemical staining of MMP-1 and TNF-α was performed in sections of cartilage (original magnification × 200 (scale bar, 400 μm) ). (K-M) ELISA measured inflammatory factorrelative factors (MMP-13, IL-6 and TNF-α) from serum. Bars with different letters are significantly different from each other at P < 0.05. (N) Immunofluorescence of TNF-α was performed in sections of chondrocytes (original magnification × 200 (scale bar, 400 μm) ).
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Figure 5. Signaling pathway research. (A) Western Blot was used to analyze the progression of the expression of PI3K/AKT, MAPK, and NF-κB signaling pathway proteins P38, p-P38, ERK, p-ERK, Akt, p-Akt, PI3K, p-PI3K, NF-κB P65, p-P65 and IκB (the gels have been run under the same experimental conditions). (B) Schematic description of relative signaling pathways that are activated by DAP. Bars with different letters are significantly different from each other at P < 0.05.
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Figure 3
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Figure 5