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NF-κB pathway in chondrocytes and ameliorates osteoarthritis progression in a rat model
International Immunopharmacology 78 (2020) 106043
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Protectin DX attenuates IL-1β-induced inflammation via the AMPK/NF-κB pathway in chondrocytes and ameliorates osteoarthritis progression in a rat model Shang Piaoa, Wei Dub, Yingliang Weia, Yue Yanga, Xinyuan Fenga, Lunhao Baia, a b
T
⁎
Department of Orthopedic Surgery, ShengJing Hospital, China Medical University, Shenyang, PR China Department of Anesthesiology, Liaoning Cancer Hospital and Institute, Shenyang, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Osteoarthritis (OA) Protectin DX (PDX) AMP-activated protein kinase (AMPK) Nuclear factor-κB (NF-κB) Chondrocyte
Protectin DX (PDX) has been reported to have extensive anti-inflammatory effects. However, it is unknown whether PDX acts as an anti-inflammatory agent in the context of osteoarthritis (OA). This study aimed to evaluate the anti-inflammatory activity of PDX in vitro and in vivo in a model of OA. Primary rat chondrocytes were preincubated with PDX 1 h prior to IL-1β treatment for 24 h. We found that PDX was nontoxic, and pretreatment with PDX increased cell viability in IL-1β-induced chondrocytes. Preincubation with PDX also efficiently inhibited the degradation of type II collagen dose-dependently. Additionally, the expression of MMP3, MMP-13, ADAMTS4, iNOS, COX-2, NO, and PGE2 decreased after IL-1β stimulation when cells were preincubated with PDX. Moreover, PDX inhibited the increase in phosphorylated NF-κB p65 and IκBα upon IL-1β stimulation, and the negative effects of IL-1β on chondrocytes were partially blocked by treatment with pyrrolidine dithiocarbamate (PDTC), a selective NF-κB inhibitor. In addition, we found that PDX increased AMPK phosphorylation in IL-1β-mediated chondrocytes. The phosphorylation of AMPK could be inhibited by compound C, a classic AMPK inhibitor. Compound C also remarkably reversed the decrease in p65 phosphorylation and MMP-13 expression caused by PDX. Furthermore, nuclear translocation of NF-κB was visible by immunofluorescence after PDX-induced AMPK activation. Additionally, we verified that PDX ameliorated cartilage degradation in monosodium iodoacetate (MIA)-induced OA rats through histological evaluation and ELISA of TNFα in the serum and intra-articular lavage fluid. In conclusion, we have shown that PDX suppresses inflammation in chondrocytes in vitro and in vivo, likely through the AMPK/NF-κB signaling pathway. Our results suggest that PDX could be a useful novel therapeutic agent for OA treatment.
1. Introduction Osteoarthritis (OA) of the knee is expected to become the fourth most common disease by 2020, which will result in an inevitable economic and social burden to patients and families [1,2]. OA is generally characterized by progressive cartilage degradation, subchondral bone hyperplasia, and synovitis; this results in pain, stiffness, and mobility loss [3]. Inflammation and inflammatory mediators play a vital role in the pathogenesis of OA. Multiple studies have shown that the synovial fluid of OA patients contains elevated levels of inflammatory mediators [4,5]. Interleukin-1β (IL-1β), a principal pro-inflammatory mediator, induces the production of matrix metalloproteinases (MMPs) and A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS), potentially resulting in down-regulation of collagen-II and
proteoglycans in articular cartilage [6,7]. Furthermore, IL-1β-stimulated chondrocytes could activate the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), triggering the release of prostaglandin E2 (PGE2) and nitric oxide (NO) [8]. Thus, additional approaches aimed at reducing the effects of inflammatory agents may provide novel therapeutics for OA. As a chronic disease of the whole knee joint, knee OA requires nonpharmacological therapies and pharmacological interventions, as well as surgical treatments, for management. Presently, the standard pharmacological treatments for OA are primarily non-steroidal anti-inflammatory drugs (NSAIDs), providing symptomatic relief [9]. However, long-term NSAIDs use may lead to gastrointestinal and cardiovascular adverse events [10,11]. This emphasizes the need to elucidate new physiological and pharmacological pathways that may be
⁎ Corresponding author at: Department of Orthopedic Surgery, ShengJing Hospital, China Medical University, Sanhao Street #36, HePing District, Shenyang, PR China. E-mail address: [email protected] (L. Bai).
https://doi.org/10.1016/j.intimp.2019.106043 Received 7 September 2019; Received in revised form 7 November 2019; Accepted 10 November 2019 1567-5769/ © 2019 The Authors. Published by Elsevier B.V. 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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of PDX for 1 h and then dosed with 10 ng/ml of IL-1β for 24 h. Then the culture medium was replaced with serum-free DMEM containing 10% CCK8 and incubated at 37 °C for 4 h. The measurements of optical densities values at 450 nm were performed using a Gen5 plate reader (BioTek, Winooski, VT, USA). The experiments were carried out in triplicate.
targeted by novel therapeutics for proper management of OA. Protectin D is derived from double lipoxygenation of docosahexaenoic acid (DHA) [12]. Protectin DX (PDX), the most widely studied member of the protectin family, has been shown to have extensive antiinflammatory properties and is effective in resolving not only acute inflammation [13], but also chronic inflammation [14,15]. The majority of studies on PDX have shown this novel agent to be closely linked with AMPK signaling [16–20]. Recent evidence from multiple studies indicates that activation of AMPK correlates to decreased nuclear translocation of NF-κB [21]. Therefore, the anti-inflammatory actions of PDX could be involved in the AMPK/NF-κB pathway. Furthermore, PDX has been shown to inhibit LPS-mediated insulin resistance and inflammatory response in adipocytes via the NF-κB pathway [17]. However, the anti-inflammatory and protective actions of PDX during OA and its underlying mechanism remain ambiguous. In this study, we aimed to examine the anti-inflammatory actions of PDX on OA in vitro and identify whether the AMPK/NF-κB signaling pathway participates in PDX′s mechanism of action. Furthermore, we investigated the effects of PDX on cartilage degeneration in vivo.
2.4. Measurement of NO, PGE2, and TNF-α The NO levels were assessed by using total nitric oxide kit (Beyotime, China) in the culture medium incubated with PDX for 1 h prior to IL-1β treatment for 24 h. The nitrate and nitrite concentrations were determined using a standard curve generated from NaNO2 afterthe Griess reaction. The production of PGE2 in the supernatants were collected and detected by enzyme-linked immunosorbent assay (ELISA) kit (R&D, USA). TNF-α levels in the intra-articular lavage fluid of the knee and in serum were measured using an ELISA kit (Boster, China). Subsequently, the protein content in intra-articular lavage fluid was measured, enabling an appropriate dilution between samples.
2. Materials and methods 2.5. Western blot analysis 2.1. Isolation and culture of primary rat chondrocytes Total proteins were extracted from the chondrocytes using RIPA lysis buffer (Beyotime, China) with 1% PMSF. The protein concentration in each sample was determined using a BCA assay kit (Beyotime, China). The proteins (30 μg) were separated by SDS-PAGE (8–10% polyacrylamide) and transferred to a PVDF membrane. Next, the PVDF membranes were blocked with 5% bovine serum albumin (BSA) (Solarbio, China) for 2 h and incubated with primary antibodies against INOS (1:500, Abcam), COX-2 (1:1000, Abcam), MMP3 (1:1000, Abcam), MMP13 (1:3000, Abcam), ADAMTS4 (1:1000, Abcam), type II collagen (1:500, Proteintech), AMPK (1:1000, Cell Signaling Technology), p-AMPK (1:1000, Cell Signaling Technology), p65 (1:1000, Cell Signaling Technology), p-p65 (1:1000, Cell Signaling Technology), IκB (1:1000, Cell Signaling Technology), p-IκB (1:1000, Cell Signaling Technology), and β-actin (1:3000, Absin) at 4 °C overnight. Finally, the membranes were incubated with a secondary antibody (1:5000, Zhongshanjinqiao) for 2 h and were visualized using enhanced chemiluminescence agents (Millipore, USA) the next day.
Knee cartilage pieces were sliced from femoral heads and knee articular cartilage tissues, which were obtained from 4-week-old SpragueDawley (SD) rats. The specimens were then incubated with pronase (2 mg/mL) (Roche, USA) for 2 h and collagenase type II (1.5 mg/mL) (Roche, USA) for 2 h. Next, isolated primary cells (P0) were resuspended in fresh Dulbecco′s modified Eagle′s medium (DMEM) (Bioind, Israel) medium supplemented with 10% fetal bovine serum (FBS) (Bioind, Israel), 1% penicillin–streptomycin solution (Bioind, Israel) in 25-mm2 flasks at 37 °C under a humidified atmosphere containing 5% CO2. Thereafter, the medium was changed every 3 days. The P0 cells, when reaching approximately 70–80% confluence, were detached continually with trypsin (Bioind, Israel) for subculture as second or third generations to prevent phenotype loss. 2.2. Pharmacological preparation and treatment Ethanol in the PDX (purity ≥ 98%) (Cayman Chemical, USA) solution was evaporated gently under vacuum, and then dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA) was immediately added as a solvent. Compound C (MCE, USA) and PDTC (MCE, USA) were dissolved in DMSO. IL-1β (Peprotech, USA) was dissolved in PBS. The drugs in DMSO were diluted in serum-free culture medium to obtain the desired concentration. As a result, the DMSO concentration in the experimental culture medium was ≤0.1% and did not result in any negative effects on cell viability. The desired concentration of PDX was determined by measuring cell viability. Additionally, the final concentrations of PDTC (10 μM) and compound C (10 μM) used were consistent with those used in previous studies [22,23]. To simulate an inflammatory response in the chondrocytes, all of the groups except the control group were dosed with 10 ng/mL of IL-1β. After preincubation with PDX and the inhibitors for 1 h and co-treatment with IL-1β for 24 h, the media supernatant and cells were collected and used for further experiments.
2.6. qRT-PCR Total RNA was extracted from the chondrocytes using TRIzol reagent (Takara, Japan), and complementary DNA (cDNA) was amplified using sequence-specific primers. RNA (1 μg) was used to synthesize cDNA using a cDNA synthesis kit (Takara, Japan). Quantitative realtime PCR was performed in an ABI Prism 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the SYBR Premix ExTaq II kit (Takara, Japan). The fold changes in relative gene expression were calculated using the 2 −ΔΔCt method, with β-actin serving as the housekeeping gene. The primers used for qPCR are shown in Table 1. 2.7. Immunofluorescence microscopy Chondrocytes were seeded onto coverslips in a 6-well plate. Once 60% confluency was reached, the supernatants were removed and replaced by serum-free medium with or without various drugs or stimulants for 24 h. After rinsing three times in PBS, the coverslips with chondrocytes were fixed with 4% paraformaldehyde for 20 min and then were permeabilized with 0.5% Triton X-100 for 20 min at room temperature. Subsequently, the cells were blocked with 5% BSA for 60 min without rinsing and were then probed with anti-p65 antibody (1:100, Cell Signaling Technology) overnight at 4 °C. On the second day, the secondary Alexa Fluor 594-conjugated antibody (1:200,
2.3. Cell viability assay PDX cytotoxicity in primary rat chondrocytes was evaluated by the CCK8 assay (Beyotime, China). Briefly, cells (1 × 104/well) were seeded onto a 96-well culture plate and treated with rising concentrations of PDX (0.5, 1, 2, 4, and 8 μM) for 24 and 48 h. The concentrations of PDX used in subsequent experiments were selected based upon these results. Next, the cells were preincubated with various concentrations 2
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Table 1 Primer sequences for qRT-PCR. TARGET GENE
FORWARD PRIMER
REVERSE PRIMER
MMP3 MMP13 ADAMTS4 COL2 β-actin
5′-TCAATCCCTCTATGGACCTCCC-3′ 5′-AAGCACCCCAAAACACCAGA-3′ 5′-CACCGAACCGACCTCTTCAA-3′ 5′-GCCAGGATGCCCGAAAATTA-3′ 5′-GATCAAGATCATTGCTCCTCCTG-3′
5′-CTGCATCGAAGGACAAAGCAG-3′ 5′-GCAGACGCCAGAAGAATCTGT-3′ 5′-GAGTTCCATCTGCCACCCGT-3′ 5′-GTCACCTCTGGGTCCTTGTTC-3′ 5′-AGGGTGTAAAACGCAGCTCA-3′
Fig. 1. Effect of PDX on primary rat chondrocyte viability and IL-1β-induced damage. PDX cytotoxicity against chondrocytes was evaluated by the CCK8 assay. Chondrocytes were incubated with PDX (0.5, 1, 2, 4, and 8 μM) for 24 or 48 h (A, B). After preincubation with PDX (1, 2, and 4 μM) for 1 h, chondrocytes were treated with IL-1β (10 ng/ml) for 24 h (C). The data are presented as the means ± SD of three independent experiments. ## p < 0.01 compared with control group. *p < 0.05, **p < 0.01 compared with IL-1β group.
Abcam) was dropped on the coverslips, and the cells were incubated for 4 h at room temperature. After rinsing three times in PBS, the chondrocytes were stained with Actin-Tracker Green (Beyotime, China) for 20 min at room temperature. Next, the chondrocytes were incubated with DAPI (Beyotime, China) for 10 min. Finally, fields selected randomly for observation were imaged using an LSM-880 confocal fluorescence microscope (Carl Zeiss, Jena, Germany).
and then transferred to 10% ethylenediamine tetraacetic acid for 6 weeks for decalcification, with the solution being replaced every 3 days. Next, the decalcified tissues were dehydrated and embedded in paraffin, and 5-μm sagittal sections were prepared for hematoxylin and eosin (H&E) and safranin O/fast green stains. The histological evaluation was conducted using the Osteoarthritis Research Society International (OARSI) scoring system in a blinded manner [24].
2.8. Animal experiments
2.11. Statistical analysis
Four-week-old male SD rats were obtained from the Animal Laboratory Center of Shengjing Hospital, China Medical University. These experiments were performed in line with the recommendations of the Ethics Committee of Shengjing Hospital, China Medical University, which approved this protocol. All rats were anesthetized by intraperitoneal injection of 1.5% pentobarbital (30 mg/kg). The knee OA model was established by intra‐articular injection of monosodium iodoacetate (MIA) (0.5 mg per joint in 30 μl of sterile saline) with a microsyringe through the lateral infrapatellar approach. Control group rats (n = 10 rats each) received an intraperitoneal injection of an equal amount of sterile saline. The OA and OA plus PDX groups were treated every 3 days with intraperitoneal injections of either PDX (10 µg/kg) or DMSO (vehicle) for 4 weeks beginning on the day after the MIA injection. Rats were sacrificed by pentobarbital overdose at 4 weeks following treatment, and knee joint specimens were isolated for further experiments.
Quantitative analysis of the Western blot bands was performed with Image J software (version 1.51; Wayne Rasband, USA). To assess the normality and homogeneity of the results, the Shapiro–Wilk and Levene tests were performed. The values are presented as the mean ± SD, and the data were analyzed using GraphPad Prism 8.0 software (San Diego, USA). One-way analysis of variance, followed by the Student′s t-test, was used to determine statistical differences. P < 0.05 was regarded as statistically significant. 3. Results 3.1. Cytotoxic effect of PDX on primary rat chondrocytes The CCK8 assay was used to determine the cytotoxicity of PDX against chondrocytes. As shown in Fig. 1A and B, no detectable cytotoxicity was observed with PDX concentrations between 0 and 4 μM. Therefore, PDX concentrations of 0, 1, 2, and 4 μM were used in future experiments. As shown in Fig. 1C, cell viability was decreased after IL1β (10 ng/ml) treatment. However, PDX was non-toxic and dose-dependently reversed IL-1β-induced cytotoxicity.
2.9. Sampling and tissue preparation Fresh blood samples were obtained instantly after the rats received pentobarbital anesthesia after 4 weeks. The serum was separated from the whole blood by centrifugation (3000 g, 4 °C, 10 min). The rat joints were dissected and were then formalin-fixed for 3 days. Intra-articular lavage fluid (IALF) was collected from the joint cavity by injection and recovery of 0.3 ml of saline 3 times using a 1 ml syringe.
3.2. PDX inhibited IL-1β-stimulated expression of NO, PGE2, iNOS, and COX-2 in chondrocytes To evaluate the anti-inflammatory potential of PDX in chondrocytes, we detected the expression of essential inflammatory cytokines COX-2, iNOS and its product, NO, and PGE2. As shown in Fig. 2A and B, NO and PGE2 production increased markedly upon stimulation with IL-1β. However, PDX treatment significantly blocked NO and
2.10. Histology After formalin-fixation for 3 days, rat joint specimens were washed 3
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Fig. 2. PDX reduced IL-1β-mediated NO, PGE2, COX-2, and iNOS expression in primary chondrocytes. Cells were incubated for 1 h with PDX (1, 2, and 4 μM) and then treated with IL-1β for 24 h. NO expression was measured using Griess reagent (A). The production of PGE2 was assessed by ELISA (B). COX-2 and iNOS were evaluated by Western blot and quantification analysis (C, D). Data are expressed as mean ± SD. All experiments were repeated in triplicate. #p < 0.05 versus control group. *p < 0.05, **p < 0.01 versus IL-1β group.
inflammatory mediator MMP-13 in chondrocytes exposed to IL-1β was evaluated. As shown in Fig. 5A and B, phosphorylation of AMPK was decreased by IL-1β, which was then enhanced by PDX. Compound C attenuated the activation of AMPK induced by PDX. Under identical conditions, phosphorylation of p65 was increased by IL-1β stimulation, which was suppressed by PDX as previously demonstrated. Compound C reversed the inhibition of NF‐κB p65 activation caused by PDX. Likewise, PDX attenuated the increase in MMP-13 caused by IL-1β, whereas compound C increased MMP-13 expression. Additionally, we performed immunofluorescence staining to explore the effect of PDX on IL-1β-mediated nuclear translocation of NF-κB p65. As shown in Fig. 5C, the majority of p65 detected was localized to the cytoplasm in control chondrocytes. IL-1β stimulation enhanced the nuclear localization of p65, and PDX blocked its translocation to the nucleus. Furthermore, compound C reversed the inhibition of nuclear translocation of p65 caused by PDX in accordance with the previous western blot results. These results suggest that PDX exerts its therapeutic effects by phosphorylating AMPK and inhibiting nuclear translocation of NF‐κB p65.
PGE2 release dose-dependently. Subsequently, we assessed the effect of PDX on iNOS and COX-2 levels by Western blot. IL-1β stimulation noticeably increased the production of iNOS and COX-2, whereas PDX treatment suppressed the production of iNOS and COX-2 in a concentration-dependent manner (Fig. 2C and D). 3.3. Effect of PDX on IL-1β-induced expression of MMP-3, MMP-13, ADAMTS-4, and collagen II production Since the crucial role of MMPs, ADAMTSs and type II collagen in the pathological feature in OA, we used RT-PCR and Western blot analysis to assess the actions of PDX on MMP-3, MMP-13, ADAMTS-4, and type II collagen levels in rat chondrocytes exposed to IL-1β. As shown in Fig. 3A–L, we found that IL-1β stimulation resulted in markedly increased levels of MMP-3, MMP-13, and ADAMTS-4 and decreased levels of collagen II expression compared to the control group. The enhanced expression of metalloproteinases and reduction of type II collagen caused by IL-1β was dose-dependently reversed by PDX pretreatment. 3.4. Effect of PDX on NF-κB signaling pathway in IL-1β-induced chondrocytes
3.6. PDX ameliorates osteoarthritis progression in a rat model
To ascertain the potential molecular mechanisms of PDX activity in chondrocytes, we used western blot analysis to evaluate alterations in the NF-κB signaling pathway. Specifically, we measured the levels of NF-κB and IκBα phosphorylation, which indicates activation of the NFκB pathway. As shown in Fig. 4A and B, the phosphorylation of NF-κB p65 and IκBα was significantly elevated by IL-1β treatment in rat chondrocytes. In addition, IL-1β stimulation lead to evident degradation of IκBα in chondrocytes. In contrast, PDX inhibited the IL-1βmediated phosphorylation of p65 and IκBα dose-dependently. Maximum inhibition of phosphorylated p65 was achieved with 4 μM PDX, in line with the previous results. This concentration was therefore used in subsequent experiments. We further elucidated whether the NF-κB signaling pathway was essential for the inhibition of IL-1β-mediated inflammation in rat chondrocytes. Treatment with PDTC, a potent NF-kB inhibitor, partially blocked the activation of NF-κB p65 and prevented IL-1β-stimulated iNOS, MMP13, and NO levels after treatment with PDX (Fig. 4C–E), indicating that PDX suppresses inflammatory mediator expression by reducing NF-κB phosphorylation.
To investigate the anti-inflammatory role of PDX against OA progression in vivo, a rat model of OA was established with MIA intraarticular injection. As shown in Fig. 6A, H&E staining and Safranin O staining confirmed that the articular cartilage in the control group presented with normal morphology and a smooth surface, while the OA group exhibited a clear reduction in the cartilage matrix, with an irregular morphological structure and rough surfaces. However, the PDX group displayed a marked increase in articular cartilage thickness and improvement in the cartilage surface, and the cartilage damage was visibly ameliorated. Similarly, as presented in Fig. 6B, the OARSI scores of the MIA-injected group were significantly higher than those of the control group, and the PDX group exhibited significantly lower OARSI scores compared to the OA group. As shown in Fig. 6C, we found that the concentrations of TNF-α in the serum of the OA group were higher than those of the control group. However, PDX treatment reduced serum TNF-α production compared to the OA group. Similar results were seen when TNF-α levels in the IALF were measured (Fig. 6D). These results indicate that PDX exerts protective actions to inhibit the OA progression in vivo.
3.5. Effect of PDX on IL-1β-mediated activation of the AMPK/NF-κB signaling pathway
4. Discussion It has been recognized that inflammation is a dominant biological event related to the pathogenesis of OA [1]. Although the precise mechanism of OA remains uncertain, a growing body of literature suggests that inflammatory factors exert a crucial action in the downstream inflammatory cascade [27]. Secreted inflammatory cytokines, such as IL1β, are primary instigators in the regional and systemic inflammatory
As a vital role in cellular regulation, AMPK is closely linked to inflammation, including inflammation in OA [25,26]. To determine whether the anti-inflammatory effect of PDX involves the AMPK/NF-κB signaling pathway, the effect of compound C, a classic AMPK inhibitor, on AMPK and p65 activation as well as the expression of the 4
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Fig. 3. PDX inhibited IL-1β-induced upregulation of MMP-3, MMP-13, ADAMTS-4, and collagen II expression in chondrocytes. Chondrocytes were incubated with PDX (1, 2, and 4 μM) for 1 h followed by IL-1β (10 ng/ml) for 24 h. mRNA levels of MMP-3, MMP-13, ADAMTS-4, and collagen II were measured by real-time PCR (AD). The expression of these proteins was evaluated by Western blot (E, F) and quantification analysis (G-J). Data are expressed as mean ± SD of triplicate values. # p < 0.05 versus control group. *p < 0.05, **p < 0.01 versus IL-1β group.
aggregation by inhibiting COX-1 and COX-2 [37]. However, few studies have examined whether PDX can exert anti-inflammatory effects in chronic degenerative diseases, such as OA. Resolvin D, another SPM family member and homologous product of double lipoxygenation of DHA, alleviates the inflammatory response in IL-1β-stimulated chondrocytes by reducing iNOS, COX-2 and MMP-13 levels, as well as NO and PGE2 production in vitro [38]. Therefore, we used an inflammatory agent to mimic OA in vitro to further demonstrate the potential cytoprotective role of PDX. As a critical inflammatory cytokine, IL-1β is associated with the pathological progression of OA; it is always detected in the synovial fluid of patients with osteoarthritis [39]. Our results indicate that IL-1β significantly decreases chondrocyte viability, which is in accordance with previous studies [40], and that PDX can boost cell viability after IL-1β treatment. These data demonstrate that PDX protects IL-1β-stimulated chondrocytes by improving cell survival. MMPs are a superfamily of proteinases that are widely present in connective tissues. MMPs are mainly in charge of the degradation and remodeling of extracellular matrix (ECM), of which type II collagen is the key component [41]. MMP-3 and MMP-13 can inhibit the synthesis of collagen and degrade components of the ECM [42,43]. Additionally, many studies have confirmed that IL-1β is co-expressed with OA-related MMPs, resulting in cartilage degradation [44,45]. Moreover,
processes of OA [28]. PDX, an endogenous DHA-derived lipid mediator, has been shown to exert potent anti-inflammatory actions [15,17,18,29]. In the present study, we demonstrated the effects of PDX on IL-1β-mediated inflammatory responses through the AMPK/NF-κB signaling pathway in primary rat chondrocytes. Given the increasing prevalence of OA and the unsatisfactory side effects of NSAIDs, it is important that novel anti-inflammatory agents are developed to relieve and control progressive inflammation. In the last decades, studies have revealed that a new array of molecules named specialized pro-resolving mediators (SPMs), which are endogenously generated from ω-3 fatty acid, function as pro-resolving mediator agonists which stimulate resolution in various ways [30]. The SPM family includes lipoxins, resolvins, protectins, and maresins [15]. Protectins have been regarded as potent agents [31]. The stereostructure and anti-inflammatory effects of protectin D1 (PD1) have been extensively investigated [32,33]. PDX, an isomer of PD1, is a recently identified double oxygenated derivative of DHA. PDX promotes the resolution of inflammation by alleviating leukocyte infiltration and boosting macrophage phagocytosis and phagocyte migration during the acute inflammatory response [15,34,35]. Morita et al. reported that PDX (mistaken as PD1 in their study) suppressed the replication of influenza virus and protected against severe infection [36]. Furthermore, PDX has been shown to reduce neutrophil activation and platelet 5
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Fig. 4. Effect of PDX on IL-1β-mediated NF-κB activation in chondrocytes. After pre-incubation with different concentrations of PDX (1, 2, and 4 μM), chondrocytes were treated with IL-1β (10 ng/ml) for 24 h. Western blots and quantification analysis were performed to assess the protein expression levels of p-p65, p65, p-IκB, and IκB (A, B). Effects of IL-1β, PDX plus IL-1β, and PDTC (10 μM) plus IL-1β on the NF-kB pathway and inflammatory responses in chondrocytes were measured. The expression levels of iNOS, MMP13, and p-p65 were significantly elevated by IL-1β treatment and were partially reversed by PDX or PDTC treatment (C, D). The Griess reagent was used to measure NO production in the supernatant (E). Data are expressed as mean ± SD of triplicate values. ##p < 0.01 versus control group. *p < 0.05, **p < 0.01, &p < 0.05 versus IL-1β group.
namely cyclooxygenase (COX) inhibitors. In fact, substantial efforts have been made to detect the desired anti-inflammatory benefits of nonselective and selective COX inhibitors. In these studies, the beneficial effects, including cell viability, degradation of enzymes, cytokine release, and NF-κB activation are in accord with our results [52]. We found that PDX at a dose as low as 4 μM in vitro exerted comparable anti-inflammatory effects to traditional COX inhibitors (10 μM) with half-dose IL-1β stimulation [53,54]. Additionally, we found that the majority of novel compounds targeting OA in the existing literature are extracts from traditional herbs. The results of reduction of inflammatory mediators (iNOS, COX2, NO, PGE2 and MMPs) and inhibition of NF-κB pathway activation by these extracts are approximately in agreement with our results [55–57]. These downstream inflammatory effectors are thought to respond to the activation of NFκB, but few studies have concentrated on specific molecular mechanisms of the upstream factors of NF-κB and further investigation is needed. AMPK has a principal role in sustaining cellular energy homeostasis and is associated with anti-inflammatory effects, anti-oxidative effects, and metabolic regulation [25,26]. White et al. found that administration of PDX caused AMPK phosphorylation and reduced insulin resistance in mouse skeletal muscle [14]. Additionally, the decrease in AMPK phosphorylation was reversed after PDX treatment, which ameliorated hepatic steatosis and gluconeogenesis by the inhibition of endoplasmic reticulum stress [19,20]. Additionally, AMPK phosphorylation after PDX treatment has been shown to suppress the activation of NF-κB p65 in palmitate-treated C2C12 cells, leading to the improvement in insulin resistance and inflammation [18]. In addition, it has been reported that PDX prevents inflammation through AMPK activation in H2O2-stimulated vascular endothelial cells [16]. On the basis of these results, we sought to determine whether the potential therapeutic actions of PDX involve the AMPK pathway in rat chondrocytes. Similarly, we revealed that PDX inhibits inflammation via AMPK-dependent signaling, which was confirmed by the suppressive effects of
chondrocytes can release aggrecanases, such as A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTs), which exert a pivotal role in OA pathophysiology [46]. IL-1β has been reported to increase ADAMTS-4 levels in chondrocytes [47]. Likewise, IL-1β can promote the production of other inflammatory factors, such as iNOS, COX-2, NO, and PGE2. In this study, we found that PDX suppressed IL1β-mediated production of MMP-3, MMP-13, and ADAMTS-4, as well as iNOS, COX-2, NO, and PGE2 dose-dependently. The results above indicate that PDX exerts a strong protective effect against the IL1βmediated inflammatory response in rat chondrocytes by inhibiting inflammatory cytokines. The NF-κB/IκB pathway is a pivotal inflammatory mechanism in OA, inducing cytokine production, cartilage degradation, and cell proliferation [48]. Under normal physiological conditions, inactive NFκB is present as a heterodimer containing the p50/p65 subunits in the cytoplasm. IκBα, a core member of the IκB inhibitory family, maintains the inactive state [49]. Once triggered by activating signals, the IκBα subunit is phosphorylated and degraded, allowing nuclear translocation of NF-κB and transcription of genes involved in ECM degradation, such as MMPs and ADAMTs [50]. Therefore, the NF-κB/IκB pathway in chondrocytes may be a potential therapeutic target for OA treatment. In our study, the nuclear translocation of NF-κB was reversed by PDX, indicating that activation of the NF-κB pathway was inhibited. The outcome is in accordance with an in vivo study which revealed that PDX is a novel inhibitor of inflammatory factors and acts through the NF-κB pathway [17]. Similarly, PD1 has been reported to suppress the activation of NF-κB in an in vivo hepatitis model [51]. To further examine whether the NF-κB pathway was associated with IL-1β-mediated inflammation in chondrocytes, we used PDTC to specifically inhibit NFκB. We found that PDTC partially blocked the activation of NF-κB exposed to IL-1β. Taken together, these outcomes indicate that PDX′s antiinflammatory properties may result from suppression of NF-κB p65. Pharmacological treatment of OA generally aims to reduce pain, the earliest symptom. This is done chiefly through the use of NSAIDs, 6
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Fig. 5. Effect of PDX on IL-1β-mediated activation of the AMPK/NF-κB signaling pathway. Chondrocytes were pre-incubated with PDX (4 μM) in the presence or absence of compound C (10 μM), followed by IL-1β treatment for 24 h. The expression levels of p-AMPK, AMPK, p-p65, p65, and MMP13 proteins were measured by Western blot analysis (A, B). The values expressed as mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01 versus control group. *p < 0.05, **p < 0.01 versus IL-1β group. $ p < 0.05, $$ p < 0.01 versus IL-1β plus PDX group. (C) Effect of PDX on nuclear translocation of p65 in IL‐1β‐induced chondrocytes. The chondrocytes were stained with anti‐p65 antibody (red) and visualized by confocal microscopy. The cytoskeleton was stained with Actin-Tracker Green (green), and the cell nucleus was identified by DAPI (blue). All experiments were repeated in triplicate. Scale bar, 50 μm.
damage. Additionally, systemic administration of PDX suppressed TNFα, a representative inflammatory cytokine, in synovial cavity fluid and serum in our OA model, further indicating a strong anti-inflammatory effect of PDX. Additionally, our previous study demonstrated that lipoxin A4, the end-product generated from ω-3 fatty acid, exerted chondroprotective effects in MIA-induced OA rats [58], which is consistent with our current results.
AMPK activation resulting from compound C. Although the specific receptor of PDX remains uncertain, current evidence indicates a strong association between PDX and AMPK [16–20]. To explore the downstream effects of AMPK activation triggered by PDX, we examined the effect of compound C on IL-1β-induced chondrocytes. The immunofluorescence staining and Western blot results showed that compound C significantly reversed the decrease in p65 phosphorylation and MMP-13 expression, which supports our hypothesis that the anti-inflammatory effects of PDX involve the AMPK/NFκB axis. Given the anti-inflammatory actions of PDX seen in vitro, we established an OA model to explore the roles of PDX on joint cartilage tissues in vivo. Morphological observations demonstrated that PDX substantially ameliorated cartilage damage and decreased the ORASI score in MIA-injected OA rats, indicating alleviation of cartilage
5. Conclusion In summary, this study showed that pre-incubation with PDX considerably attenuated inflammation in IL-1β-stimulated primary rat chondrocytes. The protective mechanism of PDX could be ascribed to the reduction of the expression of inflammatory factors via the AMPK/ 7
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Fig. 6. PDX ameliorated osteoarthritis progression in a rat model. After establishing a rat OA model by intra-articular injection of MIA, rats in the PDX group received an intraperitoneal injection of PDX (10 µg/kg) every 3 days while the OA group received DMSO (vehicle) for 4 weeks. Histological analysis was assessed by hematoxylin-eosin staining and safranin-O/fast green staining (A), and OARSI scoring (B). PDX significantly alleviated MIA-induced TNF-α production in the serum and intra-articular lavage fluid (C-D). All data are represented as mean ± SD. #p < 0.05 versus the control group, *p < 0.05 versus the OA group. Representative histologic images are shown above. [4] L.T. Nguyen, et al., Review of prospects of biological fluid biomarkers in osteoarthritis, Int. J. Mol. Sci. (2017) 18(3). [5] R. Liu-Bryan, Inflammation and intracellular metabolism: new targets in OA, Osteoarthrit. Cartil. 23 (11) (2015) 1835–1842. [6] R. Kelwick, et al., The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family, Genome Biol. 16 (2015) 113. [7] M.B. Goldring, M. Otero, Inflammation in osteoarthritis, Curr. Opin. Rheumatol. 23 (5) (2011) 471–478. [8] S.B. Abramson, et al., Nitric oxide and inflammatory mediators in the perpetuation of osteoarthritis, Curr. Rheumatol. Rep. 3 (6) (2001) 535–541. [9] D.J. Hunter, S. Bierma-Zeinstra, Osteoarthritis, Lancet 393 (10182) (2019) 1745–1759. [10] M. Atiquzzaman, et al., Role of non-steroidal anti-inflammatory drugs (NSAIDs) in the association between osteoarthritis and cardiovascular diseases: a longitudinal study, Arthrit. Rheumatol (2019). [11] M.C. Osani, et al., Duration of symptom relief and early trajectory of adverse events for oral NSAIDs in knee osteoarthritis: a systematic review and meta-analysis, Arthritis Care Res (Hoboken) (2019). [12] P. Chen, et al., Full characterization of PDX, a neuroprotectin/protectin D1 isomer, which inhibits blood platelet aggregation, FEBS Lett. 583 (21) (2009) 3478–3484. [13] X.J. Zhuo, et al., Protectin DX increases alveolar fluid clearance in rats with lipopolysaccharide-induced acute lung injury, Exp. Mol. Med. 50 (4) (2018) 49. [14] P.J. White, et al., Protectin DX alleviates insulin resistance by activating a myokineliver glucoregulatory axis, Nat. Med. 20 (6) (2014) 664–669. [15] H. Xia, et al., Protectin DX increases survival in a mouse model of sepsis by ameliorating inflammation and modulating macrophage phenotype, Sci. Rep. 7 (1) (2017) 99. [16] H.-J. Hwang, et al., Protectin DX prevents H2O2-mediated oxidative stress in vascular endothelial cells via an AMPK-dependent mechanism, Cell. Signal. 53 (2019) 14–21. [17] T.W. Jung, et al., Protectin DX attenuates LPS-induced inflammation and insulin resistance in adipocytes via AMPK-mediated suppression of the NFkappaB pathway, Am. J. Physiol. Endocrinol. Metab. (2018).
NF-κB pathway. Furthermore, we confirmed that PDX could ameliorate cartilage damage in MIA-induced OA rats. These results demonstrate that PDX may be a promising therapy for OA. Conflict of Competing Interest The authors declare that they have no conflicts of interest. Acknowledgements This study was sponsored by the National Natural Science Foundation of China (81772420, 81272050 and 31900847) Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2019.106043. References [1] E.R. Vina, C.K. Kwoh, Epidemiology of osteoarthritis: literature update, Curr. Opin. Rheumatol. 30 (2) (2018) 160–167. [2] A.D. Woolf, B. Pfleger, Burden of major musculoskeletal conditions, Bull. World Health Organ. 81 (9) (2003) 646–656. [3] V.B. Kraus, et al., Call for standardized definitions of osteoarthritis and risk stratification for clinical trials and clinical use, Osteoarthrit. Cartil. 23 (8) (2015) 1233–1241.
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[18] T.W. Jung, et al., Protectin DX ameliorates palmitate- or high-fat diet-induced insulin resistance and inflammation through an AMPK-PPARalpha-dependent pathway in mice, Sci. Rep. 7 (1) (2017) 1397. [19] T.W. Jung, et al., Protectin DX suppresses hepatic gluconeogenesis through AMPKHO-1-mediated inhibition of ER stress, Cell. Signal. 34 (2017) 133–140. [20] T.W. Jung, et al., Protectin DX ameliorates hepatic steatosis by suppression of endoplasmic reticulum stress via AMPK-induced ORP150 expression, J. Pharmacol. Exp. Ther. 365 (3) (2018) 485–493. [21] A. Salminen, J.M. Hyttinen, K. Kaarniranta, AMP-activated protein kinase inhibits NF-kappaB signaling and inflammation: impact on healthspan and lifespan, J Mol Med (Berl) 89 (7) (2011) 667–676. [22] R. Zhou, et al., Interleukin-6 enhances acid-induced apoptosis via upregulating acid-sensing ion channel 1a expression and function in rat articular chondrocytes, Int. Immunopharmacol. 29 (2) (2015) 748–760. [23] Y. Yang, et al., Mechanical stress protects against osteoarthritis via regulation of the AMPK/NF-kappaB signaling pathway, J. Cell. Physiol. 234 (6) (2019) 9156–9167. [24] K.P. Pritzker, et al., Osteoarthritis cartilage histopathology: grading and staging, Osteoarthrit. Cartil. 14 (1) (2006) 13–29. [25] C.L. Lyons, H.M. Roche, Nutritional modulation of AMPK-impact upon metabolicinflammation, Int. J. Mol. Sci. 19 (10) (2018). [26] S. Zhou, et al., AMPK deficiency in chondrocytes accelerated the progression of instability-induced and ageing-associated osteoarthritis in adult mice, Sci. Rep. 7 (2017) 43245. [27] M. Kapoor, et al., Role of proinflammatory cytokines in the pathophysiology of osteoarthritis, Nat. Rev. Rheumatol. 7 (1) (2011) 33–42. [28] X. Chevalier, T. Conrozier, P. Richette, Desperately looking for the right target in osteoarthritis: the anti-IL-1 strategy, Arthritis Res. Ther. 13 (4) (2011) 124. [29] C. Ash, Protectin and resolvin gut inflammation, Science 356 (6334) (2017) 150–151. [30] C.N. Serhan, Pro-resolving lipid mediators are leads for resolution physiology, Nature 510 (7503) (2014) 92–101. [31] M.C. Basil, B.D. Levy, Specialized pro-resolving mediators: endogenous regulators of infection and inflammation, Nat. Rev. Immunol. 16 (1) (2016) 51–67. [32] M. Aursnes, et al., Stereoselective synthesis of protectin D1: a potent anti-inflammatory and proresolving lipid mediator, Org. Biomol. Chem. 12 (3) (2014) 432–437. [33] S. Hong, et al., Resolvin D1, protectin D1, and related docosahexaenoic acid-derived products: Analysis via electrospray/low energy tandem mass spectrometry based on spectra and fragmentation mechanisms, J. Am. Soc. Mass Spectrom. 18 (1) (2007) 128–144. [34] K. Stein, et al., A role for 12/15-lipoxygenase-derived proresolving mediators in postoperative ileus: protectin DX-regulated neutrophil extravasation, J. Leukoc. Biol. 99 (2) (2016) 231–239. [35] W. Tan, et al., Protectin DX exhibits protective effects in mouse model of lipopolysaccharide-induced acute lung injury, Chin. Med. J. (Engl.) 131 (10) (2018) 1167–1173. [36] M. Morita, et al., The lipid mediator protectin D1 inhibits influenza virus replication and improves severe influenza, Cell 153 (1) (2013) 112–125. [37] M. Liu, et al., Protectin DX, a double lipoxygenase product of DHA, inhibits both ROS production in human neutrophils and cyclooxygenase activities, Lipids 49 (1) (2014) 49–57. [38] H. Benabdoune, et al., The role of resolvin D1 in the regulation of inflammatory and
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Protectin DX attenuates IL-1β-induced inflammation via the AMPK/NF-κB pathway in chondrocytes and ameliorates osteoarthritis progression in a rat model Shang Piaoa, Wei Dub, Yingliang Weia, Yue Yanga, Xinyuan Fenga, Lunhao Baia, a b
T
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Department of Orthopedic Surgery, ShengJing Hospital, China Medical University, Shenyang, PR China Department of Anesthesiology, Liaoning Cancer Hospital and Institute, Shenyang, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Osteoarthritis (OA) Protectin DX (PDX) AMP-activated protein kinase (AMPK) Nuclear factor-κB (NF-κB) Chondrocyte
Protectin DX (PDX) has been reported to have extensive anti-inflammatory effects. However, it is unknown whether PDX acts as an anti-inflammatory agent in the context of osteoarthritis (OA). This study aimed to evaluate the anti-inflammatory activity of PDX in vitro and in vivo in a model of OA. Primary rat chondrocytes were preincubated with PDX 1 h prior to IL-1β treatment for 24 h. We found that PDX was nontoxic, and pretreatment with PDX increased cell viability in IL-1β-induced chondrocytes. Preincubation with PDX also efficiently inhibited the degradation of type II collagen dose-dependently. Additionally, the expression of MMP3, MMP-13, ADAMTS4, iNOS, COX-2, NO, and PGE2 decreased after IL-1β stimulation when cells were preincubated with PDX. Moreover, PDX inhibited the increase in phosphorylated NF-κB p65 and IκBα upon IL-1β stimulation, and the negative effects of IL-1β on chondrocytes were partially blocked by treatment with pyrrolidine dithiocarbamate (PDTC), a selective NF-κB inhibitor. In addition, we found that PDX increased AMPK phosphorylation in IL-1β-mediated chondrocytes. The phosphorylation of AMPK could be inhibited by compound C, a classic AMPK inhibitor. Compound C also remarkably reversed the decrease in p65 phosphorylation and MMP-13 expression caused by PDX. Furthermore, nuclear translocation of NF-κB was visible by immunofluorescence after PDX-induced AMPK activation. Additionally, we verified that PDX ameliorated cartilage degradation in monosodium iodoacetate (MIA)-induced OA rats through histological evaluation and ELISA of TNFα in the serum and intra-articular lavage fluid. In conclusion, we have shown that PDX suppresses inflammation in chondrocytes in vitro and in vivo, likely through the AMPK/NF-κB signaling pathway. Our results suggest that PDX could be a useful novel therapeutic agent for OA treatment.
1. Introduction Osteoarthritis (OA) of the knee is expected to become the fourth most common disease by 2020, which will result in an inevitable economic and social burden to patients and families [1,2]. OA is generally characterized by progressive cartilage degradation, subchondral bone hyperplasia, and synovitis; this results in pain, stiffness, and mobility loss [3]. Inflammation and inflammatory mediators play a vital role in the pathogenesis of OA. Multiple studies have shown that the synovial fluid of OA patients contains elevated levels of inflammatory mediators [4,5]. Interleukin-1β (IL-1β), a principal pro-inflammatory mediator, induces the production of matrix metalloproteinases (MMPs) and A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS), potentially resulting in down-regulation of collagen-II and
proteoglycans in articular cartilage [6,7]. Furthermore, IL-1β-stimulated chondrocytes could activate the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), triggering the release of prostaglandin E2 (PGE2) and nitric oxide (NO) [8]. Thus, additional approaches aimed at reducing the effects of inflammatory agents may provide novel therapeutics for OA. As a chronic disease of the whole knee joint, knee OA requires nonpharmacological therapies and pharmacological interventions, as well as surgical treatments, for management. Presently, the standard pharmacological treatments for OA are primarily non-steroidal anti-inflammatory drugs (NSAIDs), providing symptomatic relief [9]. However, long-term NSAIDs use may lead to gastrointestinal and cardiovascular adverse events [10,11]. This emphasizes the need to elucidate new physiological and pharmacological pathways that may be
⁎ Corresponding author at: Department of Orthopedic Surgery, ShengJing Hospital, China Medical University, Sanhao Street #36, HePing District, Shenyang, PR China. E-mail address: [email protected] (L. Bai).
https://doi.org/10.1016/j.intimp.2019.106043 Received 7 September 2019; Received in revised form 7 November 2019; Accepted 10 November 2019 1567-5769/ © 2019 The Authors. Published by Elsevier B.V. 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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of PDX for 1 h and then dosed with 10 ng/ml of IL-1β for 24 h. Then the culture medium was replaced with serum-free DMEM containing 10% CCK8 and incubated at 37 °C for 4 h. The measurements of optical densities values at 450 nm were performed using a Gen5 plate reader (BioTek, Winooski, VT, USA). The experiments were carried out in triplicate.
targeted by novel therapeutics for proper management of OA. Protectin D is derived from double lipoxygenation of docosahexaenoic acid (DHA) [12]. Protectin DX (PDX), the most widely studied member of the protectin family, has been shown to have extensive antiinflammatory properties and is effective in resolving not only acute inflammation [13], but also chronic inflammation [14,15]. The majority of studies on PDX have shown this novel agent to be closely linked with AMPK signaling [16–20]. Recent evidence from multiple studies indicates that activation of AMPK correlates to decreased nuclear translocation of NF-κB [21]. Therefore, the anti-inflammatory actions of PDX could be involved in the AMPK/NF-κB pathway. Furthermore, PDX has been shown to inhibit LPS-mediated insulin resistance and inflammatory response in adipocytes via the NF-κB pathway [17]. However, the anti-inflammatory and protective actions of PDX during OA and its underlying mechanism remain ambiguous. In this study, we aimed to examine the anti-inflammatory actions of PDX on OA in vitro and identify whether the AMPK/NF-κB signaling pathway participates in PDX′s mechanism of action. Furthermore, we investigated the effects of PDX on cartilage degeneration in vivo.
2.4. Measurement of NO, PGE2, and TNF-α The NO levels were assessed by using total nitric oxide kit (Beyotime, China) in the culture medium incubated with PDX for 1 h prior to IL-1β treatment for 24 h. The nitrate and nitrite concentrations were determined using a standard curve generated from NaNO2 afterthe Griess reaction. The production of PGE2 in the supernatants were collected and detected by enzyme-linked immunosorbent assay (ELISA) kit (R&D, USA). TNF-α levels in the intra-articular lavage fluid of the knee and in serum were measured using an ELISA kit (Boster, China). Subsequently, the protein content in intra-articular lavage fluid was measured, enabling an appropriate dilution between samples.
2. Materials and methods 2.5. Western blot analysis 2.1. Isolation and culture of primary rat chondrocytes Total proteins were extracted from the chondrocytes using RIPA lysis buffer (Beyotime, China) with 1% PMSF. The protein concentration in each sample was determined using a BCA assay kit (Beyotime, China). The proteins (30 μg) were separated by SDS-PAGE (8–10% polyacrylamide) and transferred to a PVDF membrane. Next, the PVDF membranes were blocked with 5% bovine serum albumin (BSA) (Solarbio, China) for 2 h and incubated with primary antibodies against INOS (1:500, Abcam), COX-2 (1:1000, Abcam), MMP3 (1:1000, Abcam), MMP13 (1:3000, Abcam), ADAMTS4 (1:1000, Abcam), type II collagen (1:500, Proteintech), AMPK (1:1000, Cell Signaling Technology), p-AMPK (1:1000, Cell Signaling Technology), p65 (1:1000, Cell Signaling Technology), p-p65 (1:1000, Cell Signaling Technology), IκB (1:1000, Cell Signaling Technology), p-IκB (1:1000, Cell Signaling Technology), and β-actin (1:3000, Absin) at 4 °C overnight. Finally, the membranes were incubated with a secondary antibody (1:5000, Zhongshanjinqiao) for 2 h and were visualized using enhanced chemiluminescence agents (Millipore, USA) the next day.
Knee cartilage pieces were sliced from femoral heads and knee articular cartilage tissues, which were obtained from 4-week-old SpragueDawley (SD) rats. The specimens were then incubated with pronase (2 mg/mL) (Roche, USA) for 2 h and collagenase type II (1.5 mg/mL) (Roche, USA) for 2 h. Next, isolated primary cells (P0) were resuspended in fresh Dulbecco′s modified Eagle′s medium (DMEM) (Bioind, Israel) medium supplemented with 10% fetal bovine serum (FBS) (Bioind, Israel), 1% penicillin–streptomycin solution (Bioind, Israel) in 25-mm2 flasks at 37 °C under a humidified atmosphere containing 5% CO2. Thereafter, the medium was changed every 3 days. The P0 cells, when reaching approximately 70–80% confluence, were detached continually with trypsin (Bioind, Israel) for subculture as second or third generations to prevent phenotype loss. 2.2. Pharmacological preparation and treatment Ethanol in the PDX (purity ≥ 98%) (Cayman Chemical, USA) solution was evaporated gently under vacuum, and then dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA) was immediately added as a solvent. Compound C (MCE, USA) and PDTC (MCE, USA) were dissolved in DMSO. IL-1β (Peprotech, USA) was dissolved in PBS. The drugs in DMSO were diluted in serum-free culture medium to obtain the desired concentration. As a result, the DMSO concentration in the experimental culture medium was ≤0.1% and did not result in any negative effects on cell viability. The desired concentration of PDX was determined by measuring cell viability. Additionally, the final concentrations of PDTC (10 μM) and compound C (10 μM) used were consistent with those used in previous studies [22,23]. To simulate an inflammatory response in the chondrocytes, all of the groups except the control group were dosed with 10 ng/mL of IL-1β. After preincubation with PDX and the inhibitors for 1 h and co-treatment with IL-1β for 24 h, the media supernatant and cells were collected and used for further experiments.
2.6. qRT-PCR Total RNA was extracted from the chondrocytes using TRIzol reagent (Takara, Japan), and complementary DNA (cDNA) was amplified using sequence-specific primers. RNA (1 μg) was used to synthesize cDNA using a cDNA synthesis kit (Takara, Japan). Quantitative realtime PCR was performed in an ABI Prism 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the SYBR Premix ExTaq II kit (Takara, Japan). The fold changes in relative gene expression were calculated using the 2 −ΔΔCt method, with β-actin serving as the housekeeping gene. The primers used for qPCR are shown in Table 1. 2.7. Immunofluorescence microscopy Chondrocytes were seeded onto coverslips in a 6-well plate. Once 60% confluency was reached, the supernatants were removed and replaced by serum-free medium with or without various drugs or stimulants for 24 h. After rinsing three times in PBS, the coverslips with chondrocytes were fixed with 4% paraformaldehyde for 20 min and then were permeabilized with 0.5% Triton X-100 for 20 min at room temperature. Subsequently, the cells were blocked with 5% BSA for 60 min without rinsing and were then probed with anti-p65 antibody (1:100, Cell Signaling Technology) overnight at 4 °C. On the second day, the secondary Alexa Fluor 594-conjugated antibody (1:200,
2.3. Cell viability assay PDX cytotoxicity in primary rat chondrocytes was evaluated by the CCK8 assay (Beyotime, China). Briefly, cells (1 × 104/well) were seeded onto a 96-well culture plate and treated with rising concentrations of PDX (0.5, 1, 2, 4, and 8 μM) for 24 and 48 h. The concentrations of PDX used in subsequent experiments were selected based upon these results. Next, the cells were preincubated with various concentrations 2
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Table 1 Primer sequences for qRT-PCR. TARGET GENE
FORWARD PRIMER
REVERSE PRIMER
MMP3 MMP13 ADAMTS4 COL2 β-actin
5′-TCAATCCCTCTATGGACCTCCC-3′ 5′-AAGCACCCCAAAACACCAGA-3′ 5′-CACCGAACCGACCTCTTCAA-3′ 5′-GCCAGGATGCCCGAAAATTA-3′ 5′-GATCAAGATCATTGCTCCTCCTG-3′
5′-CTGCATCGAAGGACAAAGCAG-3′ 5′-GCAGACGCCAGAAGAATCTGT-3′ 5′-GAGTTCCATCTGCCACCCGT-3′ 5′-GTCACCTCTGGGTCCTTGTTC-3′ 5′-AGGGTGTAAAACGCAGCTCA-3′
Fig. 1. Effect of PDX on primary rat chondrocyte viability and IL-1β-induced damage. PDX cytotoxicity against chondrocytes was evaluated by the CCK8 assay. Chondrocytes were incubated with PDX (0.5, 1, 2, 4, and 8 μM) for 24 or 48 h (A, B). After preincubation with PDX (1, 2, and 4 μM) for 1 h, chondrocytes were treated with IL-1β (10 ng/ml) for 24 h (C). The data are presented as the means ± SD of three independent experiments. ## p < 0.01 compared with control group. *p < 0.05, **p < 0.01 compared with IL-1β group.
Abcam) was dropped on the coverslips, and the cells were incubated for 4 h at room temperature. After rinsing three times in PBS, the chondrocytes were stained with Actin-Tracker Green (Beyotime, China) for 20 min at room temperature. Next, the chondrocytes were incubated with DAPI (Beyotime, China) for 10 min. Finally, fields selected randomly for observation were imaged using an LSM-880 confocal fluorescence microscope (Carl Zeiss, Jena, Germany).
and then transferred to 10% ethylenediamine tetraacetic acid for 6 weeks for decalcification, with the solution being replaced every 3 days. Next, the decalcified tissues were dehydrated and embedded in paraffin, and 5-μm sagittal sections were prepared for hematoxylin and eosin (H&E) and safranin O/fast green stains. The histological evaluation was conducted using the Osteoarthritis Research Society International (OARSI) scoring system in a blinded manner [24].
2.8. Animal experiments
2.11. Statistical analysis
Four-week-old male SD rats were obtained from the Animal Laboratory Center of Shengjing Hospital, China Medical University. These experiments were performed in line with the recommendations of the Ethics Committee of Shengjing Hospital, China Medical University, which approved this protocol. All rats were anesthetized by intraperitoneal injection of 1.5% pentobarbital (30 mg/kg). The knee OA model was established by intra‐articular injection of monosodium iodoacetate (MIA) (0.5 mg per joint in 30 μl of sterile saline) with a microsyringe through the lateral infrapatellar approach. Control group rats (n = 10 rats each) received an intraperitoneal injection of an equal amount of sterile saline. The OA and OA plus PDX groups were treated every 3 days with intraperitoneal injections of either PDX (10 µg/kg) or DMSO (vehicle) for 4 weeks beginning on the day after the MIA injection. Rats were sacrificed by pentobarbital overdose at 4 weeks following treatment, and knee joint specimens were isolated for further experiments.
Quantitative analysis of the Western blot bands was performed with Image J software (version 1.51; Wayne Rasband, USA). To assess the normality and homogeneity of the results, the Shapiro–Wilk and Levene tests were performed. The values are presented as the mean ± SD, and the data were analyzed using GraphPad Prism 8.0 software (San Diego, USA). One-way analysis of variance, followed by the Student′s t-test, was used to determine statistical differences. P < 0.05 was regarded as statistically significant. 3. Results 3.1. Cytotoxic effect of PDX on primary rat chondrocytes The CCK8 assay was used to determine the cytotoxicity of PDX against chondrocytes. As shown in Fig. 1A and B, no detectable cytotoxicity was observed with PDX concentrations between 0 and 4 μM. Therefore, PDX concentrations of 0, 1, 2, and 4 μM were used in future experiments. As shown in Fig. 1C, cell viability was decreased after IL1β (10 ng/ml) treatment. However, PDX was non-toxic and dose-dependently reversed IL-1β-induced cytotoxicity.
2.9. Sampling and tissue preparation Fresh blood samples were obtained instantly after the rats received pentobarbital anesthesia after 4 weeks. The serum was separated from the whole blood by centrifugation (3000 g, 4 °C, 10 min). The rat joints were dissected and were then formalin-fixed for 3 days. Intra-articular lavage fluid (IALF) was collected from the joint cavity by injection and recovery of 0.3 ml of saline 3 times using a 1 ml syringe.
3.2. PDX inhibited IL-1β-stimulated expression of NO, PGE2, iNOS, and COX-2 in chondrocytes To evaluate the anti-inflammatory potential of PDX in chondrocytes, we detected the expression of essential inflammatory cytokines COX-2, iNOS and its product, NO, and PGE2. As shown in Fig. 2A and B, NO and PGE2 production increased markedly upon stimulation with IL-1β. However, PDX treatment significantly blocked NO and
2.10. Histology After formalin-fixation for 3 days, rat joint specimens were washed 3
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Fig. 2. PDX reduced IL-1β-mediated NO, PGE2, COX-2, and iNOS expression in primary chondrocytes. Cells were incubated for 1 h with PDX (1, 2, and 4 μM) and then treated with IL-1β for 24 h. NO expression was measured using Griess reagent (A). The production of PGE2 was assessed by ELISA (B). COX-2 and iNOS were evaluated by Western blot and quantification analysis (C, D). Data are expressed as mean ± SD. All experiments were repeated in triplicate. #p < 0.05 versus control group. *p < 0.05, **p < 0.01 versus IL-1β group.
inflammatory mediator MMP-13 in chondrocytes exposed to IL-1β was evaluated. As shown in Fig. 5A and B, phosphorylation of AMPK was decreased by IL-1β, which was then enhanced by PDX. Compound C attenuated the activation of AMPK induced by PDX. Under identical conditions, phosphorylation of p65 was increased by IL-1β stimulation, which was suppressed by PDX as previously demonstrated. Compound C reversed the inhibition of NF‐κB p65 activation caused by PDX. Likewise, PDX attenuated the increase in MMP-13 caused by IL-1β, whereas compound C increased MMP-13 expression. Additionally, we performed immunofluorescence staining to explore the effect of PDX on IL-1β-mediated nuclear translocation of NF-κB p65. As shown in Fig. 5C, the majority of p65 detected was localized to the cytoplasm in control chondrocytes. IL-1β stimulation enhanced the nuclear localization of p65, and PDX blocked its translocation to the nucleus. Furthermore, compound C reversed the inhibition of nuclear translocation of p65 caused by PDX in accordance with the previous western blot results. These results suggest that PDX exerts its therapeutic effects by phosphorylating AMPK and inhibiting nuclear translocation of NF‐κB p65.
PGE2 release dose-dependently. Subsequently, we assessed the effect of PDX on iNOS and COX-2 levels by Western blot. IL-1β stimulation noticeably increased the production of iNOS and COX-2, whereas PDX treatment suppressed the production of iNOS and COX-2 in a concentration-dependent manner (Fig. 2C and D). 3.3. Effect of PDX on IL-1β-induced expression of MMP-3, MMP-13, ADAMTS-4, and collagen II production Since the crucial role of MMPs, ADAMTSs and type II collagen in the pathological feature in OA, we used RT-PCR and Western blot analysis to assess the actions of PDX on MMP-3, MMP-13, ADAMTS-4, and type II collagen levels in rat chondrocytes exposed to IL-1β. As shown in Fig. 3A–L, we found that IL-1β stimulation resulted in markedly increased levels of MMP-3, MMP-13, and ADAMTS-4 and decreased levels of collagen II expression compared to the control group. The enhanced expression of metalloproteinases and reduction of type II collagen caused by IL-1β was dose-dependently reversed by PDX pretreatment. 3.4. Effect of PDX on NF-κB signaling pathway in IL-1β-induced chondrocytes
3.6. PDX ameliorates osteoarthritis progression in a rat model
To ascertain the potential molecular mechanisms of PDX activity in chondrocytes, we used western blot analysis to evaluate alterations in the NF-κB signaling pathway. Specifically, we measured the levels of NF-κB and IκBα phosphorylation, which indicates activation of the NFκB pathway. As shown in Fig. 4A and B, the phosphorylation of NF-κB p65 and IκBα was significantly elevated by IL-1β treatment in rat chondrocytes. In addition, IL-1β stimulation lead to evident degradation of IκBα in chondrocytes. In contrast, PDX inhibited the IL-1βmediated phosphorylation of p65 and IκBα dose-dependently. Maximum inhibition of phosphorylated p65 was achieved with 4 μM PDX, in line with the previous results. This concentration was therefore used in subsequent experiments. We further elucidated whether the NF-κB signaling pathway was essential for the inhibition of IL-1β-mediated inflammation in rat chondrocytes. Treatment with PDTC, a potent NF-kB inhibitor, partially blocked the activation of NF-κB p65 and prevented IL-1β-stimulated iNOS, MMP13, and NO levels after treatment with PDX (Fig. 4C–E), indicating that PDX suppresses inflammatory mediator expression by reducing NF-κB phosphorylation.
To investigate the anti-inflammatory role of PDX against OA progression in vivo, a rat model of OA was established with MIA intraarticular injection. As shown in Fig. 6A, H&E staining and Safranin O staining confirmed that the articular cartilage in the control group presented with normal morphology and a smooth surface, while the OA group exhibited a clear reduction in the cartilage matrix, with an irregular morphological structure and rough surfaces. However, the PDX group displayed a marked increase in articular cartilage thickness and improvement in the cartilage surface, and the cartilage damage was visibly ameliorated. Similarly, as presented in Fig. 6B, the OARSI scores of the MIA-injected group were significantly higher than those of the control group, and the PDX group exhibited significantly lower OARSI scores compared to the OA group. As shown in Fig. 6C, we found that the concentrations of TNF-α in the serum of the OA group were higher than those of the control group. However, PDX treatment reduced serum TNF-α production compared to the OA group. Similar results were seen when TNF-α levels in the IALF were measured (Fig. 6D). These results indicate that PDX exerts protective actions to inhibit the OA progression in vivo.
3.5. Effect of PDX on IL-1β-mediated activation of the AMPK/NF-κB signaling pathway
4. Discussion It has been recognized that inflammation is a dominant biological event related to the pathogenesis of OA [1]. Although the precise mechanism of OA remains uncertain, a growing body of literature suggests that inflammatory factors exert a crucial action in the downstream inflammatory cascade [27]. Secreted inflammatory cytokines, such as IL1β, are primary instigators in the regional and systemic inflammatory
As a vital role in cellular regulation, AMPK is closely linked to inflammation, including inflammation in OA [25,26]. To determine whether the anti-inflammatory effect of PDX involves the AMPK/NF-κB signaling pathway, the effect of compound C, a classic AMPK inhibitor, on AMPK and p65 activation as well as the expression of the 4
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Fig. 3. PDX inhibited IL-1β-induced upregulation of MMP-3, MMP-13, ADAMTS-4, and collagen II expression in chondrocytes. Chondrocytes were incubated with PDX (1, 2, and 4 μM) for 1 h followed by IL-1β (10 ng/ml) for 24 h. mRNA levels of MMP-3, MMP-13, ADAMTS-4, and collagen II were measured by real-time PCR (AD). The expression of these proteins was evaluated by Western blot (E, F) and quantification analysis (G-J). Data are expressed as mean ± SD of triplicate values. # p < 0.05 versus control group. *p < 0.05, **p < 0.01 versus IL-1β group.
aggregation by inhibiting COX-1 and COX-2 [37]. However, few studies have examined whether PDX can exert anti-inflammatory effects in chronic degenerative diseases, such as OA. Resolvin D, another SPM family member and homologous product of double lipoxygenation of DHA, alleviates the inflammatory response in IL-1β-stimulated chondrocytes by reducing iNOS, COX-2 and MMP-13 levels, as well as NO and PGE2 production in vitro [38]. Therefore, we used an inflammatory agent to mimic OA in vitro to further demonstrate the potential cytoprotective role of PDX. As a critical inflammatory cytokine, IL-1β is associated with the pathological progression of OA; it is always detected in the synovial fluid of patients with osteoarthritis [39]. Our results indicate that IL-1β significantly decreases chondrocyte viability, which is in accordance with previous studies [40], and that PDX can boost cell viability after IL-1β treatment. These data demonstrate that PDX protects IL-1β-stimulated chondrocytes by improving cell survival. MMPs are a superfamily of proteinases that are widely present in connective tissues. MMPs are mainly in charge of the degradation and remodeling of extracellular matrix (ECM), of which type II collagen is the key component [41]. MMP-3 and MMP-13 can inhibit the synthesis of collagen and degrade components of the ECM [42,43]. Additionally, many studies have confirmed that IL-1β is co-expressed with OA-related MMPs, resulting in cartilage degradation [44,45]. Moreover,
processes of OA [28]. PDX, an endogenous DHA-derived lipid mediator, has been shown to exert potent anti-inflammatory actions [15,17,18,29]. In the present study, we demonstrated the effects of PDX on IL-1β-mediated inflammatory responses through the AMPK/NF-κB signaling pathway in primary rat chondrocytes. Given the increasing prevalence of OA and the unsatisfactory side effects of NSAIDs, it is important that novel anti-inflammatory agents are developed to relieve and control progressive inflammation. In the last decades, studies have revealed that a new array of molecules named specialized pro-resolving mediators (SPMs), which are endogenously generated from ω-3 fatty acid, function as pro-resolving mediator agonists which stimulate resolution in various ways [30]. The SPM family includes lipoxins, resolvins, protectins, and maresins [15]. Protectins have been regarded as potent agents [31]. The stereostructure and anti-inflammatory effects of protectin D1 (PD1) have been extensively investigated [32,33]. PDX, an isomer of PD1, is a recently identified double oxygenated derivative of DHA. PDX promotes the resolution of inflammation by alleviating leukocyte infiltration and boosting macrophage phagocytosis and phagocyte migration during the acute inflammatory response [15,34,35]. Morita et al. reported that PDX (mistaken as PD1 in their study) suppressed the replication of influenza virus and protected against severe infection [36]. Furthermore, PDX has been shown to reduce neutrophil activation and platelet 5
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Fig. 4. Effect of PDX on IL-1β-mediated NF-κB activation in chondrocytes. After pre-incubation with different concentrations of PDX (1, 2, and 4 μM), chondrocytes were treated with IL-1β (10 ng/ml) for 24 h. Western blots and quantification analysis were performed to assess the protein expression levels of p-p65, p65, p-IκB, and IκB (A, B). Effects of IL-1β, PDX plus IL-1β, and PDTC (10 μM) plus IL-1β on the NF-kB pathway and inflammatory responses in chondrocytes were measured. The expression levels of iNOS, MMP13, and p-p65 were significantly elevated by IL-1β treatment and were partially reversed by PDX or PDTC treatment (C, D). The Griess reagent was used to measure NO production in the supernatant (E). Data are expressed as mean ± SD of triplicate values. ##p < 0.01 versus control group. *p < 0.05, **p < 0.01, &p < 0.05 versus IL-1β group.
namely cyclooxygenase (COX) inhibitors. In fact, substantial efforts have been made to detect the desired anti-inflammatory benefits of nonselective and selective COX inhibitors. In these studies, the beneficial effects, including cell viability, degradation of enzymes, cytokine release, and NF-κB activation are in accord with our results [52]. We found that PDX at a dose as low as 4 μM in vitro exerted comparable anti-inflammatory effects to traditional COX inhibitors (10 μM) with half-dose IL-1β stimulation [53,54]. Additionally, we found that the majority of novel compounds targeting OA in the existing literature are extracts from traditional herbs. The results of reduction of inflammatory mediators (iNOS, COX2, NO, PGE2 and MMPs) and inhibition of NF-κB pathway activation by these extracts are approximately in agreement with our results [55–57]. These downstream inflammatory effectors are thought to respond to the activation of NFκB, but few studies have concentrated on specific molecular mechanisms of the upstream factors of NF-κB and further investigation is needed. AMPK has a principal role in sustaining cellular energy homeostasis and is associated with anti-inflammatory effects, anti-oxidative effects, and metabolic regulation [25,26]. White et al. found that administration of PDX caused AMPK phosphorylation and reduced insulin resistance in mouse skeletal muscle [14]. Additionally, the decrease in AMPK phosphorylation was reversed after PDX treatment, which ameliorated hepatic steatosis and gluconeogenesis by the inhibition of endoplasmic reticulum stress [19,20]. Additionally, AMPK phosphorylation after PDX treatment has been shown to suppress the activation of NF-κB p65 in palmitate-treated C2C12 cells, leading to the improvement in insulin resistance and inflammation [18]. In addition, it has been reported that PDX prevents inflammation through AMPK activation in H2O2-stimulated vascular endothelial cells [16]. On the basis of these results, we sought to determine whether the potential therapeutic actions of PDX involve the AMPK pathway in rat chondrocytes. Similarly, we revealed that PDX inhibits inflammation via AMPK-dependent signaling, which was confirmed by the suppressive effects of
chondrocytes can release aggrecanases, such as A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTs), which exert a pivotal role in OA pathophysiology [46]. IL-1β has been reported to increase ADAMTS-4 levels in chondrocytes [47]. Likewise, IL-1β can promote the production of other inflammatory factors, such as iNOS, COX-2, NO, and PGE2. In this study, we found that PDX suppressed IL1β-mediated production of MMP-3, MMP-13, and ADAMTS-4, as well as iNOS, COX-2, NO, and PGE2 dose-dependently. The results above indicate that PDX exerts a strong protective effect against the IL1βmediated inflammatory response in rat chondrocytes by inhibiting inflammatory cytokines. The NF-κB/IκB pathway is a pivotal inflammatory mechanism in OA, inducing cytokine production, cartilage degradation, and cell proliferation [48]. Under normal physiological conditions, inactive NFκB is present as a heterodimer containing the p50/p65 subunits in the cytoplasm. IκBα, a core member of the IκB inhibitory family, maintains the inactive state [49]. Once triggered by activating signals, the IκBα subunit is phosphorylated and degraded, allowing nuclear translocation of NF-κB and transcription of genes involved in ECM degradation, such as MMPs and ADAMTs [50]. Therefore, the NF-κB/IκB pathway in chondrocytes may be a potential therapeutic target for OA treatment. In our study, the nuclear translocation of NF-κB was reversed by PDX, indicating that activation of the NF-κB pathway was inhibited. The outcome is in accordance with an in vivo study which revealed that PDX is a novel inhibitor of inflammatory factors and acts through the NF-κB pathway [17]. Similarly, PD1 has been reported to suppress the activation of NF-κB in an in vivo hepatitis model [51]. To further examine whether the NF-κB pathway was associated with IL-1β-mediated inflammation in chondrocytes, we used PDTC to specifically inhibit NFκB. We found that PDTC partially blocked the activation of NF-κB exposed to IL-1β. Taken together, these outcomes indicate that PDX′s antiinflammatory properties may result from suppression of NF-κB p65. Pharmacological treatment of OA generally aims to reduce pain, the earliest symptom. This is done chiefly through the use of NSAIDs, 6
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Fig. 5. Effect of PDX on IL-1β-mediated activation of the AMPK/NF-κB signaling pathway. Chondrocytes were pre-incubated with PDX (4 μM) in the presence or absence of compound C (10 μM), followed by IL-1β treatment for 24 h. The expression levels of p-AMPK, AMPK, p-p65, p65, and MMP13 proteins were measured by Western blot analysis (A, B). The values expressed as mean ± SD of three independent experiments. #p < 0.05, ##p < 0.01 versus control group. *p < 0.05, **p < 0.01 versus IL-1β group. $ p < 0.05, $$ p < 0.01 versus IL-1β plus PDX group. (C) Effect of PDX on nuclear translocation of p65 in IL‐1β‐induced chondrocytes. The chondrocytes were stained with anti‐p65 antibody (red) and visualized by confocal microscopy. The cytoskeleton was stained with Actin-Tracker Green (green), and the cell nucleus was identified by DAPI (blue). All experiments were repeated in triplicate. Scale bar, 50 μm.
damage. Additionally, systemic administration of PDX suppressed TNFα, a representative inflammatory cytokine, in synovial cavity fluid and serum in our OA model, further indicating a strong anti-inflammatory effect of PDX. Additionally, our previous study demonstrated that lipoxin A4, the end-product generated from ω-3 fatty acid, exerted chondroprotective effects in MIA-induced OA rats [58], which is consistent with our current results.
AMPK activation resulting from compound C. Although the specific receptor of PDX remains uncertain, current evidence indicates a strong association between PDX and AMPK [16–20]. To explore the downstream effects of AMPK activation triggered by PDX, we examined the effect of compound C on IL-1β-induced chondrocytes. The immunofluorescence staining and Western blot results showed that compound C significantly reversed the decrease in p65 phosphorylation and MMP-13 expression, which supports our hypothesis that the anti-inflammatory effects of PDX involve the AMPK/NFκB axis. Given the anti-inflammatory actions of PDX seen in vitro, we established an OA model to explore the roles of PDX on joint cartilage tissues in vivo. Morphological observations demonstrated that PDX substantially ameliorated cartilage damage and decreased the ORASI score in MIA-injected OA rats, indicating alleviation of cartilage
5. Conclusion In summary, this study showed that pre-incubation with PDX considerably attenuated inflammation in IL-1β-stimulated primary rat chondrocytes. The protective mechanism of PDX could be ascribed to the reduction of the expression of inflammatory factors via the AMPK/ 7
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Fig. 6. PDX ameliorated osteoarthritis progression in a rat model. After establishing a rat OA model by intra-articular injection of MIA, rats in the PDX group received an intraperitoneal injection of PDX (10 µg/kg) every 3 days while the OA group received DMSO (vehicle) for 4 weeks. Histological analysis was assessed by hematoxylin-eosin staining and safranin-O/fast green staining (A), and OARSI scoring (B). PDX significantly alleviated MIA-induced TNF-α production in the serum and intra-articular lavage fluid (C-D). All data are represented as mean ± SD. #p < 0.05 versus the control group, *p < 0.05 versus the OA group. Representative histologic images are shown above. [4] L.T. Nguyen, et al., Review of prospects of biological fluid biomarkers in osteoarthritis, Int. J. Mol. Sci. (2017) 18(3). [5] R. Liu-Bryan, Inflammation and intracellular metabolism: new targets in OA, Osteoarthrit. Cartil. 23 (11) (2015) 1835–1842. [6] R. Kelwick, et al., The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family, Genome Biol. 16 (2015) 113. [7] M.B. Goldring, M. Otero, Inflammation in osteoarthritis, Curr. Opin. Rheumatol. 23 (5) (2011) 471–478. [8] S.B. Abramson, et al., Nitric oxide and inflammatory mediators in the perpetuation of osteoarthritis, Curr. Rheumatol. Rep. 3 (6) (2001) 535–541. [9] D.J. Hunter, S. Bierma-Zeinstra, Osteoarthritis, Lancet 393 (10182) (2019) 1745–1759. [10] M. Atiquzzaman, et al., Role of non-steroidal anti-inflammatory drugs (NSAIDs) in the association between osteoarthritis and cardiovascular diseases: a longitudinal study, Arthrit. Rheumatol (2019). [11] M.C. Osani, et al., Duration of symptom relief and early trajectory of adverse events for oral NSAIDs in knee osteoarthritis: a systematic review and meta-analysis, Arthritis Care Res (Hoboken) (2019). [12] P. Chen, et al., Full characterization of PDX, a neuroprotectin/protectin D1 isomer, which inhibits blood platelet aggregation, FEBS Lett. 583 (21) (2009) 3478–3484. [13] X.J. Zhuo, et al., Protectin DX increases alveolar fluid clearance in rats with lipopolysaccharide-induced acute lung injury, Exp. Mol. Med. 50 (4) (2018) 49. [14] P.J. White, et al., Protectin DX alleviates insulin resistance by activating a myokineliver glucoregulatory axis, Nat. Med. 20 (6) (2014) 664–669. [15] H. Xia, et al., Protectin DX increases survival in a mouse model of sepsis by ameliorating inflammation and modulating macrophage phenotype, Sci. Rep. 7 (1) (2017) 99. [16] H.-J. Hwang, et al., Protectin DX prevents H2O2-mediated oxidative stress in vascular endothelial cells via an AMPK-dependent mechanism, Cell. Signal. 53 (2019) 14–21. [17] T.W. Jung, et al., Protectin DX attenuates LPS-induced inflammation and insulin resistance in adipocytes via AMPK-mediated suppression of the NFkappaB pathway, Am. J. Physiol. Endocrinol. Metab. (2018).
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