Part 1:Acteoside Counteracts Interleukin-1β-Induced Catabolic Processes Through The Modulation Of Mitogen-Activated Protein Kinases And The NFκB Cellular Signaling Pathway
Mar 05, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
HyangI Lim ,1 Do Kyung Kim ,1 Tae-Hyeon Kim ,1 Kyeong-Rok Kang
Osteoarthritis (OA) is the most common degenerative joint disease with chronic joint pain caused by progressive degeneration of articular cartilage at synovial joints. Acteoside, a caffeoylphenylethanoid glycoside, has various biological activities such as antimicrobial, anti-inflammatory, anticancer, antioxidative, cytoprotective, and neuroprotective effect. Further, oral administration of acteoside at a high dosage does not cause genotoxicity. Therefore, the aim of the present study is to verify the anticatabolic effects of acteoside against osteoarthritis and its anticatabolic signaling pathway. Acteoside did not decrease the viabilities of mouse fibroblast L929 cells used as normal cells and primary rat chondrocytes. Acteoside counteracted the IL-1β-induced proteoglycan loss in the chondrocytes and articular cartilage by suppressing the expression and activation of a cartilage-degrading enzyme such as matrix metalloproteinase- (MMP-) 13, MMP-1, and MMP-3. Furthermore, acteoside suppressed the expression of inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase-2, nitric oxide, and prostaglandin E2 in the primary rat chondrocytes treated with IL-1β. Subsequently, the expression of proinflammatory cytokines was decreased by acteoside in the primary rat chondrocytes treated with IL-1β. Moreover, acteoside suppressed not only the phosphorylation of mitogen-activated protein kinases in primary rat chondrocytes treated with IL-1β but also the translocation of NFκB from the cytosol to the nucleus through suppression of its phosphorylation. Oral administration of 5 and 10mg/kg acteoside attenuated the progressive degeneration of articular cartilage in the osteoarthritic mouse model generated by destabilization of the medial meniscus. Our findings indicate that acteoside is a promising potential anticatabolic agent or supplement to attenuate or prevent progressive degeneration of articular cartilage.
1. Introduction
Osteoarthritis (OA) is the most common degenerative joint disease with chronic joint pain caused by progressive degeneration of articular cartilage at synovial joints [1]. Due to the increase in life expectancy, the prevalence of OA with loss of mobility and chronic joint pain caused by progressive degeneration of articular cartilage at synovial joints is estimated to be 18% and 9.6% in women after menopause and in men, respectively [2]. Although the 2 Oxidative Medicine and Cellular Longevity worldwide prevalence of OA increases annually, the pathophysiological etiology of OA is still unknown. It may be caused by very complex and multifactorial risk factors such as aging, gender, genetic inheritance, traumatic joint injury, and severe mechanical joint load. Furthermore, the neuro- pathological relationships between progressive degeneration of articular cartilage and the development of chronic joint pain are unknown [3]. Hence, the goal of clinical management for patients with OA is the maintenance of body mobility and mechanical joint function through relief from chronic joint pain, using pharmacological and nonpharmacological approaches and joint replacement surgery. The demand for the development of effective intervention or supplementation, with long-term biological safety, to prevent or attenuate OA to maintain life quality through maintenance of mechanical joint function in the elderly population is increasing.
As shown in Figure 1, acteoside (CAS No. 61276-17-3; C29H36O15) is a caffeoylphenylethanoid glycoside isolated from several herbal plants such as Verbascum chlorides [4], Buddleja globosa [5], and Plantago australis [6]. Acteoside has various biological activities such as antimicrobial [5], anti-inflammatory [7], anticancer [8], antioxidative [9], cytoprotective [9], and neuroprotective effect [10]. Further, oral administration of acteoside at a high dosage does not cause genotoxicity [11].
Hence, we hypothesized that acteoside with anti- inflammatory biological safety has anticatabolic effects associated with the protection of articular cartilage against progressive degeneration of articular cartilage through suppression of catabolic factors such as the proinflammatory cytokines, inflammatory mediators, and cartilage-degrading enzymes in synovial joints. Therefore, the aim of this study was to investigate the acteoside-induced anticatabolic effects and its cellular signaling pathway both in vitro, using primary chondrocytes isolated from the articular cartilage of rat knee joint, and in vivo, using an OA animal model generated by surgical destabilization of the medial meniscus in the knee joint of mice.
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2. Methods
2.1. Cell Culture. Primary rat chondrocytes were isolated from the articular cartilage of rat (5-day-old; Sprague–Daw- ley) knee joints, in accordance with the protocol (CIA- CUC2019-A0027) approved by the Institutional Animal Care and Use Committee of Chosun University, Gwangju, Republic of Korea. Isolated primary rat chondrocytes were maintained in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12) (Thermo Scientific, Rock- ford, IL, USA) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Grand Island, NY, USA), antibiotics (50U/mL penicillin and 50μg/mL streptomycin), and 50μg/mL ascorbic acid. The normal mouse fibroblast L-929 cell line was purchased from American Type Culture Collection (ATCC). According to the instructions provided from ATCC, L-929 cells were cultured in the Eagle’s minimum essential medium, containing 10% FBS, and were grown in a humidified incubator at 37°C with 5% CO2.
2.2. Cell Viability Assay. The dimethyl thiazolyl diphenyltetrazolium salt (MTT) assay was performed to assess the viabilities of mouse fibroblast cell line L929 cells used as a normal cell and primary rat chondrocytes treated with acteoside. Briefly, L929 cells, and primary rat chondrocytes were cultured at a cell density of 8×105 cells/mL in culture plates for 24h and then treated with 2.5, 5, 10, 25, 50, and 100μM acteoside for 24h. After treatment with MTT solution, both L929 cells and chondrocytes were further cultured for 4h. After incubation, the formed MTT crystals were suspended completely in dimethyl sulfoxide and measured for absorbance at 570nm using a spectrometer (Epoch microplate spectrophotometer, BioTek®, Winooski, VT, USA) to assess cell viability.
2.3. Cell Live/Dead Assay. Cell survival was performed using Cell Live/Dead assay kit (Molecular Probes, Carlsbad, CA, USA), which is composed of green calcein-AM for labeling live cells (with green fluorescence) and ethidium homodimer-1 for labeling dead cells (with red fluorescence). Briefly, both L929 cells and primary rat chondrocytes were cultured at a cell density of 8×105 cells/mL on chamber slides (Nunc® Lab-Tek® Chamber Slide™ system; Sigma- Aldrich; Merck KGaA) for 24h and then treated with 50 and 100μM acteoside for 24h. After cultivation, a cell survival assay was performed according to the manufacturer’s instructions. Thereafter, stained cells were imaged using a fluorescence microscope (Eclipse TE200; Nikon Instruments, Melville, NY).
2.4. Dimethylmethylene Blue (DMMB) Assay. DMMB assay was performed to assess the alteration of proteoglycan content in primary rat chondrocytes treated with acteoside for 21 days in the presence or absence of IL-1β. To maintain the characteristics of primary rat chondrocytes for 21 days, primary rat chondrocytes (2×106 cells) were suspended in 1mL of 1.2% alginate and then encapsulated by dripping the cell/alginate suspension to a solution of 105mM CaCl2. The primary rat chondrocytes encapsulated in alginate were cultured for 24h in DMEM/F12 (containing 10% FBS, 50U/mL penicillin, 50μg/mL streptomycin, and 50μg/mL ascorbic acid) and then adapted for 24h in DMEM/F12 containing 1% mini-insulin–transferrin–selenium (mini-ITS) and 50μg/mL ascorbic acid. Subsequently, the chondrocytes were treated with 50 or 100μM acteoside in the presence or absence of 1ng/mL IL-1β for 21 days. On day 21, the primary rat chondrocytes were collected for assessment of proteoglycan content using the DMMB assay, as described previously [12]. In addition, to quantify proteoglycan content per cell and assess the proliferation of primary rat chondrocytes, cell numbers were measured by DNA assay using PicoGreen (Molecular Probes, Carlsbad, CA), according to the manufacturer’s instructions.
2.5. Ex Vivo Organ Culture of Rat Articular Cartilage Tissues. Articular cartilage tissues were isolated from the knee joints of 5-day-old Sprague–Dawley rats and then cultured in DMEM/F12 supplemented with 10% FBS. Next, the articular cartilage
samples were treated with 100μM acteoside in the presence or absence of 10ng/mL IL-1β for 7 days. At the end of the culture period, the samples were collected and fixed in 4% paraformaldehyde for 72h for histological analysis.
2.6. Histological Analysis. Histological analysis using safranin-O and fast green staining was performed to verify proteoglycan loss in the articular cartilage treated with 100μM acteoside in the presence or absence of 10ng/mL IL-1β for 7 days. Briefly, fixed articular cartilage samples were decalcified in ethylenediaminetetraacetic acid and then embedded in paraffin. Thereafter, the prepared paraffin blocks containing articular cartilage were serially sliced to 5μm thickness and placed on slides. Safranin-O and fast green staining was subsequently performed to assess proteo- glycan loss in the articular cartilage ground substance. In addition, hematoxylin and eosin staining was performed to observe the general morphology of the articular cartilage.
2.7. Western Blotting. Western blotting was performed to investigate the expression of catabolic factors including MMP-13, MMP-1, MMP-3, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) and the alteration of cellular signaling molecules such as mitogen-activated protein kinases and nuclear factor-kappa B (NFκB). Briefly, rat primary chondrocytes were treated with 50 or 100μM acteoside in the presence or absence of 10ng/mL IL-1β for 24h. Thereafter, rat primary chondrocytes were harvested by centrifugation and were lysed using lysis buffer (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions. In addition, to verify the nuclear translocation of NFκB, rat primary chondrocytes were treated with 50 or 100μM acteoside in the presence or absence of 10ng/mL IL-1β for 24h. Thereafter, cytosolic and nuclear fractions were extracted using NE-PER™ Nuclear and Cytoplasmic extraction reagents (Thermo Sci- entific, Rockford, IL, USA) according to the manufacturer’s instructions. The concentration of total protein extracted from primary rat chondrocytes was determined using a bicinchoninic acid protein assay kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. In addition, the conditioned medium was collected to detect the levels of cartilage-degrading enzymes secreted from chondrocytes. Equal amounts of protein and conditioned medium were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes. Thereafter, western blotting was performed using targeted primary anti-bodies against MMP-13, MMP-1, MMP-3, iNOS, COX-2, phosphoERK1/2, total-ERK1/2, phosphop38, total-p38, phosphor JNK, total JNK, phosphoNFκB, total NFκB, β- actin, and lamin B. Immunoreactive bands were visualized using an enhanced chemiluminescence system (Thermo Sci- entific, Rockford, IL, USA) according to the manufacturer’s instruction and then imaged by a Microchemi device (DNR Bioimaging Systems, Jerusalem, Israel).
2.8. Quantitative Polymerase Chain Reaction (qPCR) and Quantitative Real-Time PCR (qRT-PCR). Primary rat chondrocytes were treated with 50 or 100μM acteoside in the presence or absence of 10ng/mL IL-1β for 24h. Thereafter, total RNA was isolated from the primary rat chondrocytes using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA concentration was measured using a NanoDrop 2000 (Thermo Scientific, Rockford, IL, USA). To synthesize cDNA, 1μg RNA was reverse transcribed using a ThermoScript reverse transcription-PCR system (Invitro- gen, Carlsbad, CA, USA) according to the manufacturer’s instructions. qPCR of cDNA was performed using 2× TOPsimple™ DyeMIX-Taq (Enzynomics, Seoul, Republic of Korea) and specific primers on a TaKaRa PCR Thermal Cycler Device (TaKaRa Bio Inc., Shiga, Japan). Thereafter, the PCR products were electrophoresed on an agarose gel to determine the expression levels of target genes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. In addition, for qRT-PCR, cDNA was amplified using an Eco™ Real-Time PCR system (illumine Inc., San Diego, CA, USA). β-Actin was used as an endogenous control. The sequences of the primers used in the qPCR and qRT-PCR are summarized in Tables 1 and 2, respectively.
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3. Results
3.1. Acteoside Does Not Affect L929 Cell and Primary Rat Chondrocyte Viability. The mouse fibroblast cell line L929 used as normal cells was treated with 2.5, 5, 10, 25, 50, and 100μM acteoside for 24h. Thereafter, the MTT assay was performed to assess the cytotoxicity of acteoside on L929 cells. As shown in Figure 2(a), relative viabilities of L929 cells were determined to be 94:8±8%, 93:6±7%, 100 ± 5%, 103:9± 5%, 126:1±8%, and 122:7±4% at 2.5, 5, 10, 25, 50, and 100μM acteoside, respectively, compared with control (100:02 ± 3%). Furthermore, to verify the cytotoxicity of acteoside on primary rat chondrocytes, the MTT assay was performed as shown in Figure 2(b). The viabilities of primary rat chondrocytes treated with 2.5, 5, 10, 25, 50, and 100μM acteoside were determined as 114 ± 4%, 117:8±6%, 123:9± 5%, 132:6±4%, 153:1± 7%, and 142:1±6%, respectively, compared with control (100:4±5%). Furthermore, to confirm the effect of acteoside on the viability of both L929 cells and primary rat chondrocytes, a Cell Live/Dead assay was performed as shown in Figure 2(c). The number of dead cells stained as red fluorescence did not increase for both L929 cells and primary rat chondrocytes treated with 50 and 100μM acteoside for 24h. These data consistently demonstrated that defined dosage of acteoside did not affect the viability of L929 cells and primary rat chondrocytes. Thus,50μM acteoside and 100μM acteoside, which are nontoxic doses in both L929 cells and primary rat chondrocytes, were used to verify its anticatabolic effects in vitro studies using primary rat chondrocytes.
3.2. Acteoside Counteracts IL-1β-Induced Proteoglycan Loss in Primary Rat Chondrocytes. Primary rat chondrocytes embedded in alginate beads were treated with 50 and 100μM acteoside in the presence or absence of 1ng/mL IL- 1β for 21 days. Thereafter, a DMMB assay was performed to assess the alteration in proteoglycan content as shown in Figure 3(a). The relative proteoglycan contents were determined as 88:3±18:1% and 86:8±16:3% in the primary rat chondrocytes treated with 50 and 100μM acteoside, respectively, compared with control (103:8±32:3%). Although the relative proteoglycan contents were decreased by acteoside, these results were not significant. However, the relative proteoglycan content significantly decreased by 37:1± 14:7% in the primary rat chondrocytes treated with 1ng/mL IL-1β, but 50 and 100μM acteoside significantly reduced the proteoglycan content by 57 ± 12:4% and 64 ± 14:5%, respectively, in the presence of 1ng/mL IL-1β. Subsequently, to verify whether acteoside suppresses the IL-1β- induced proteoglycan loss, articular cartilage dissected from rat knee joints was treated with 100μM acteoside in the presence or absence of 10ng/mL IL-1β for 7 days. Thereafter, histological assessments using H&E staining and safranin-O and fast green staining were performed as shown in Figure 3(b). The morphological alteration was not observed using H&E staining; however, safranin-O and fast green staining revealed that the proteoglycan stained as the red color did not alter in the articular cartilages treated with 100μM acteoside compared with that in control. However, severe proteoglycan loss was induced by 10ng/mLIL-1β in the articular cartilage, and 100μM acteoside significantly suppressed the proteoglycan loss in the articular cartilage treated with 10ng/mL IL- 1β. Collectively, these data consistently show that acteoside has an anticatabolic effect that retards the degeneration of articular cartilage through counteracting IL-1β-induced proteoglycan loss.
3.3. Acteoside Has an Anticatabolic Effect That Suppresses MMP Expression and Activation in Primary Rat Chondrocytes Treated with IL-1β. To investigate whether acteoside-induced anticatabolic effect is associated with the suppression of MMP expression and activation, primary rat chondrocytes were treated with 50 and 100μM acteoside in the presence or absence of 10ng/mL IL-1β for 24h. Thereafter, the alterations in MMPs were investigated. As shown in Figure 4(a), although the expression of cartilage-degrading enzymes such as MMP-13, MMP-1, and MMP-3 was significantly increased in the conditioned media of primary rat chondrocytes treated with 10ng/mL IL-1β, it was decreased by acteoside in a dose-dependent manner. Furthermore, results of both qPCR (Figure 4(b)) and qRT-PCR (Figure 4(c)) revealed that IL-1β significantly increased the mRNA levels of MMPs such as MMP-13, MMP-1, and MMP-3 in the primary rat chondrocytes. However, they decreased dose-dependently in the primary rat chondrocytes treated with 50 and 100μM acteoside. Moreover, 50 and 100μM acteoside effectively suppressed the activation of MMPs in the rat primary chondrocytes treated with 10ng/mL IL-1β (Figure 4(d)). Taken together, these data consistently indicate that acteoside has an anticatabolic effect that suppresses the expression and activation of cartilage-degrading enzymes.
3.4. Acteoside Suppresses the Expression and Production of IL1β-Induced Catabolic Inflammatory Mediators and Proinflammatory Cytokines in Primary Rat Chondrocytes. To determine whether acteoside has a preventive effect against OA, primary rat chondrocytes were treated with 50 and 100μM acteoside in the presence or absence of 10ng/mL IL-1β for 24h. Thereafter, the alterations in inflammatory mediators, representative catabolic factors such as iNOS, COX-2, and PGE2, were investigated. The mRNA levels of iNOS, COX-2, and PTGS-2 were significantly increased by IL-1β in the primary rat chondrocytes.
However, they decreased dose-dependently in the primary rat chondrocytes treated with acteoside (Figure 5(a)). Furthermore, acteoside not only suppressed the expression of iNOS and COX-2 in the primary rat chondrocytes treated with IL-1β (Figure 5(b)) but also significantly decreased the relative production of NO and PGE2 as shown in Figures 5(c) and 5(d), respectively. These data suggest that acteoside suppresses the expression of inflammatory mediator-induced proinflammatory cytokines that act as catabolic factors to induce the progressive degeneration of articular cartilage. Hence, to investigate the expressional alteration of proinflammatory cytokines by 50μM acteoside in the primary rat chondrocytes treated with 10ng/mL IL- 1β, a cytokine array was performed as shown in Figure 6. Acteoside suppressed the expression of cytokine-induced neutrophil chemoattractant- (CINC-) 2, CINC-3, ciliary neurotrophic factor (CNTF), fractalkine (CX3CL1), IL-1α, IL- 1β, leptin, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein- (MIP-) 3α, and β-nerve growth factor (NGF) in the primary rat chondrocytes treated with IL-1β compared with IL-1β alone. Taken together, these data suggest consistently that acteoside prevents the progressive degeneration of articular cartilage through suppression of inflammatory mediators and proinflammatory cytokines against the IL-1β-induced catabolic effects in primary rat chondrocytes.
3.5. Acteoside Suppresses MAPK and NFκB Phosphorylation in Primary Rat Chondrocytes Treated with IL-1β. To investigate the cellular signaling pathways associated with acteoside-induced anticatabolic effects against proinflamma- tory cytokine IL-1β alteration of MAPK and NFκB, primary rat chondrocytes were treated with 50 and 100μM acteoside in the presence or absence of IL-1β for 24h. Thereafter, total protein was extracted and electrophoresed on the SDS-PAGE gel to perform the western blot. As shown in Figure 7, MAPK such as ERK1/2, p38, and JNK were significantly phosphorylated in the primary rat chondrocytes treated with IL-1β, whereas 50 and 100μM acteoside did not significantly induce the phosphorylation of MAPK compared to the control in primary rat chondrocyte. However, 50 and 100μM acteoside dose-dependently suppressed the IL-1β-induced MAPK phosphorylation in primary rat chondrocytes. Furthermore, the phosphorylation of NFκB in the primary rat chondrocytes treated with 10ng/mL IL-1β was gradually decreased by acteoside in a dose-dependent manner. These data indicate that MAPK and NFκB cellular signaling pathways are closely involved with the acteoside-induced anticatabolic effects against IL-1β in primary rat chondrocytes.
3.6. Acteoside Suppresses Translocation of NFκB from the Cytosol to the Nucleus through Suppression of IL-1β-Induced NFκB Phosphorylation in Primary Rat Chondrocytes. To investigate whether acteoside suppresses the translocation of NFκB from the cytosol to the nucleus, primary rat chondrocytes were treated with 50 and 100μg/mL acteoside in the presence or absence of 10ng/mL IL-1β. As shown in Figure 8(a), NFκB was significantly translocated to the nucleus from the cytosol of the primary rat chondrocytes treated with 10ng/mL IL-1β. However, it was significantly inhibited by acteoside in a dose-dependent manner. Furthermore, although the NFκB level was increased in the nuclear fraction extracted from the primary rat chondrocytes treated with 10ng/mL IL-1β, it was dose-dependently decreased by acteoside as shown in Figure 8(b). NFκB level was decreased in the cytosolic fraction extracted from the primary rat chondrocytes treated with 10ng/mL IL-1β, but it was dose-dependently increased by acteoside. Taken together, these data consistently indicate that acteoside-induced anticatabolic effects against IL-1β are involved in the suppression of translocation from the cytosol to the nucleus on the modulation of the NFκB signaling pathway in primary rat chondrocytes.
3.7. Acteoside Attenuates Progressive Degeneration of Articular Cartilage in the Surgical DMM-Induced Knee Joint OA Animals. To elucidate the acteoside-induced anticatabolic effects in vivo, OA-induced animals generated by the surgical DMM performed on the knee joint of BALB/c mice were orally administrated 5 and 10mg/kg acteoside resolved in 5% ethanol every other day for 8 weeks. Thereafter, knee joints were histologically assessed using safranin-O and fast green staining as shown in Figure 9. The proteoglycan loss and injury of the articular cartilage surface were significantly increased in the knee joint dissected from DMM-induced OA animals. However, the oral administration of acteoside suppressed the proteoglycan loss and less injury of articular cartilage compared with vehicle only. Furthermore, Mankin’s grading score was significantly increased in the OA animal group (n =5, 3± 0:7) supplied vehicle only compared with Naïve (n =5, 0:67 ± 0:5). However, the oral administration of 5 and 10mg/kg acteoside into the OA animal group (n =5) decreased the Mankin grading score by 2±0:7 and 1:67 ± 0:5, respectively, compared with vehicle only. Taken together, these data indicate that the oral administration of acteoside attenuates the progressive degeneration of articular cartilage in synovial joints with catabolic conditions.
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