| BackgroudOsteoarthritis (OA) is a multiple joint destruction as the main feature of progressive degeneration disease, is a common disease prevalence in the medial ages and elderly, the main characteristics are articular cartilage damage, the decrease synthesis of the extracellular matrix, the subchondral bone changes and synovial inflammation, etc.Worldwide,epidemiology shows that more than 10% of people over the age of 60 patients with osteoarthritis, this health problem usually be underestimated. Characteristic changes of OA on X-ray examination are osteophyte formation and sclerosis of subchondral bone lesions. Osteoarthritis is associated unknown etiology, can be contributed by many factors, its clinical presentation is not the same, patients with symptoms not severe only show the knee joint pain, initially is paroxysmal, afterwards is persistent, the pain is worse after activities, particularly when up and down the stairs. In recent years, numerous researches were conducted to investigate on the etiology of OA, but the underlying pathogenesis is not fully clarified. OA used to be considered the sole consequence of any process leading to increased pressure on one particular joint (e.g., overload on weight-bearing joints, anatomical joint incongruency) or fragility of cartilage matrix (genetic alterations of matrix components). This paradigm was mainly based on the observation that chondrocytes, the only cell type present in cartilage, have very low metabolism activity with no ability to repair cartilage. Progress in molecular biology in the 1990s has profoundly modified this paradigm. The discovery that many soluble mediators such as cytokines or prostaglandins can increase the production of matrix metalloproteinases (MMPs) by chondrocytes led to the first steps of an "inflammatory" theory.Thus, initially considered cartilage driven, OA is a much more complex disease with inflammatory mediators released by cartilage, bone and synovium.IL-1β as a proinflammatory factor, plays a pivotal role in the pathogenesis of OA, as well as induced the inflammatory process and apoptosis. IL-1β is produced by mononuclear cells, osteoblasts andsynovial tissues, induce the production of a number of inflammatory and catabolic factors, secret a series of inflammatory factors and proteolytic enzymes. In articular cells, IL-1β amplify and perpetuate the OA disease process by inducing the production of other proinflammatory cytokines such as IL-6 and chemokines such as IL-8, it is able to along with other cytokines active inflammatory process. In patients with OA, levels of both IL-1β are elevated in the synovial fluid, synovial membrane, subchondral bone and cartilage. After intra-articular injections of interleukin-1, all joint cartilages of rabbit showed a more severe damage than that injection of other cytokines. It show thatproteoglycan (glycosaminoglycan) lost extensively and Glycosaminoglycan biosynthesiswas initially depressed, but at one week had almost doubled its rate as compared with the normal side. The biological activation of cells by IL-1β is mediated through its specific cell-surface receptor IL1 receptor type I (IL-1R I). Expression of this receptor is increased on human OA chondrocytes and synovial fibroblasts in comparison with normal cells. IL-1 only found at the surface of cartilage with a few chondrocytes, after stimulation by trauma and free radicals, IL-1 beta receptors is over-expressed, high expression of IL-1 beta receptors bind to membrane of chondrocytes, through the transmission system, which interfere with the normal metabolism of chondrocytes, change normal structure and function of chondrocytes, promote cartilage inflammation and induce apoptosis, lead to degradation of cartilage matrix and participate in synovial inflammatory lesions, affect bone metabolism. It is one of the essential cytokines produced locally from OA chondrocytes that have been shown to upregulate the production of matrix degrading enzymes and to inhibit proteoglycan synthesis, resulting in loss of cartilage. The effect of IL-1β on cartilage homeostasis was induced by MMPs over-expression and activation that lead to extracellular matrix ECM degradation. Up-regulation of mRNA, especially increase synthesis of MMP-3, MMP-9, MMP-13, is able to disturb the balance of matrix metalloproteinase (MMPs)-tissue inhibitor of metalloproteinase (TIMPs), resulted in anabolic macromolecules degradation.MMPs can specificity of cracked collagen molecules, which leads to the damage of collagen network, decrease the stress resistance of the articular cartilage, induce apoptosis of chondrocytes and extracellular matrix degradation, eventually cartilage was destructed. COX-2 is a rate-limiting enzyme for generation of prostaglandin E2 (PGE2) metabolites, an important mediator of inflammation and the anabolic/catabolic process linked to OA [6]. iNOS has been demonstrated to catalyze the oxidation to generate amounts of nitric oxide (NO)within the joint fluid of osteoarthritis chondrocytes, leading to cartilage destruction. NO contributes to articular inflammation and destruction by enhancing the activation and production of MMPs, inhibiting the synthesis of anabolic macromolecules such as collagen and proteoglycan, inhibiting the production of IL-1Ra, and promoting chondrocyte apoptosis.IL-1β also induces the production of reactive oxygen species (ROS)which contribute to cartilage degradation. Consequently, high level of IL-1β stimulate the production the NO and ROS, accelerate the apoptosis of chondrocytes, associated with articular joint degeneration and dysfunction.Commonly prescribed OA medications include NSAIDs, analgesic drugs, locally administered corticosteroids and viscosupplementation.In clinical department, mainstream pharmacological treatments of OA are non-steroidal anti-inflammatory drugs (NSAIDs). However, traditional NSAIDs are associated with high costs and various side effects, such as serious upper gastrointestinal complications like bleeding and perforation. Therefore, it is a considerable clinical interest to identify a novel compound extracted from natural plants, as an alternative or adjunct to conventional therapy for OAAucubin, a natural compound has been found in various plants including leaves of Aucuba japonica and Eucommia ulmoides, proves to possess a variety of pharmacological effects, notably, anti-inflammatory effect. One investigation confirms that Aucubin extracted from Castilleja tenuiflora, a Mexican medicinal plant, inhibits the histamine and bradykinin production from ear edema induced by arachidonic acid and carrageenan. Recent evidence has shown that Aucubin possesses anti-inflammatory property and inhibits NF-κB activation. Moreover, an interesting study addressing the pharmaceutical effects of Eucommia ulmoides, a native Chinese medicinal herb whose major ingredient is Aucubin, indicates that it can inhibit inflammatory factors and prevents the degradation of the cartilage matrix in rats with OA.It is an urgent request to identify a novel compound extracted from natural plants with minimal side effect and high efficiency, as an alternative or adjunct to conventional therapy for OA. The present study was aim to investigate the anti-inflammatory and chondro-protective role, as well as the biosafety, of Aucubin at the cellular level, provide an evidence to a natural compound extract from plants on its clinical therapeutic potential for alternative management of OA.Methods1. Chondrocyte isolation and cultureNormal articular cartilage was isolated from the knees and femoral heads of 4-week-old Sprague-Dawley (SD) rats under sterile conditions. Well-preserved rat cartilage was washed with PBS containing penicillin/streptomycin solution five times, and then cut into small pieces. Cartilage pieces were digested primarily with 0.25% Trypsin-EDTA Solution for 30 min and subsequently with 0.2% collagenase type II in Dulbecco’s modified Eagle’ smedium(DMEM) for 2 h at 37℃. The released articular chondrocytes were resuspended in DMEMcontaining 10% FBS and seeded onto a 25 cm2 culture flask at 37℃ in a humidified atmosphere of 5% CO2-95% air. Up to approximately 80-90% confluency, chondrocytes were passaged at a ratio of 1:3. To avoid the phenotype loss, passages 1-3 chondrocytes were used in our study.2. Chondrocytes identificationMicroscopy:in the chondrocytes isolation, culture and different period’s generation, microscope was used to observe the morphology, adherence and growth, growth characteristics and the density of cells. The photos were taken on the original generation, one generation, two generation, and respectively.Alcian blue staining:Wash cells with PBS solution gently to remove all solution. Then the chondrocytes were fixed with 4% paraformaldehyde 30 min. Add 1% of alcian blue solution, staining over night, washed with DH2O several times, until the colorless, get a microscope photographs.Immunocytochemistry collagen Ⅱ:Drops one to three generation of cells on the cover glass, use climbing technique, after being growth into a single basic cells, remove the cover glass, fixed with 4% paraformaldehyde for 20 min,incubation with 3% hydrogen peroxide for 10 min at room temperature, afterwards washing with PBS buffer three times, each time for 5 min, add the normal immune animal serum incubation for 10 min at room temperature.Remove serum, add 1:100 rabbit anti-collagen type Ⅱ,4℃ for the night. Wash 3 times with PBS buffer, each time a 5 min. Second antibody was applied, incubation for 10 min at room temperature, PBS buffer washing three times, each time 5min.DAB chromogenic, hematoxylin multicolor, anhydrous ethanol dehydration, sealed.The microscope was used to observed and photo record.3. Cell viability assayThe cytotoxicity of Aucubin to chondrocytes was determined by CCK8. Briefly, rat chondrocytes (1×104/well) in 96-well plates were pretreated first without and then with Aucubin (1,10,20,50 μM) for 24 h and 48 h respectively. At the indicated time,10 μl of the CCK-8 solution was added to each well of the plat and incubated for another 2 h at 37℃. Cell viability in each group was calculated using the value of OD All experiments were performed in triplicate.4. Effects of Aucubin on inflammatory and Catabolic activities in IL-1β-induced ChondrocytesRat chondrocytes cultured in 6-well plates were pretreated first without and then with Aucubin (1,10,20,50 μM) for 24 h. The interaction of NO in a system was measured by determination of total nitrate and nitrite concentrations in the samples. Nitrite concentration was measured by the Griess reagent and absorbance was determined at 540 nm. Real-Time PCR and western blot were used to test expression of MMP-3、 MMP-9、MMP-13、iNOS、COX-2 on chondrocytes stimulated by IL-1β in the protein and gene levels respectively. The present study further explored molecular mechanisms and involved signaling pathway of the inhibited effect of Aucubin on IL-1β induced inflammatory responds and apoptosis.5. Time Dependent Effect of IL-1β on NF-κBTo examine the phosphorylation timing of the NF-κB signaling, the phosphorylation levels of NF-κB p65, IκBα and IKKα/β in IL-1β stimulated chondrocytes were assessed by western blot at various time points(0、0.5、1、2、6h).6. Effect of Aucubin on IL-1β-Induced activation of NF-κBRat chondrocytes cultured in 6-well plates were pretreated first without and then with Aucubin (1,10,20,50 μM) for 2 h.To detect the phosphorylation of the NF-κB signaling, the phosphorylation levels of NF-κB p65, IκBα and IKKα/β in IL-1β stimulated chondrocytes were assessed by Western blotting. Immunofluorescence microscopy was employed to reveal translocation of p65 subunit of NF-κB from the cytoplasm to the nucleus in chondrocytes in response to NF-κB activation by IL-1β.7. Effects of Aucubin on elevation ROS in IL-1β-induced ChondrocytesRat chondrocytes cultured in confocal dish were pretreated first without and then with Aucubin (1,10,20,50 μM) for 30min.The DCFH-DA were used to test whether Aucubin is able to inhibit the elevation of ROS in IL-1β-induced Chondrocytes.8. Effects of Aucubin on apoptosis in IL-1β-induced ChondrocytesRat chondrocytes cultured in 6-well plates were pretreated first without and then with Aucubin (1,10,20,50 μM) for 24 h. apoptosis was quantified by Flow Cytometry(FCM) using AnnexinV staining or Propidium Iodide staining. Western blot were used to identified the early stage of chondrocytes of apoptosis. Furturemore, the underlying molecular mechanisms were detected by scavenging ROS.9. Effects ofAucubin on IL-1β-induced apoptosis-related protein in ChondrocytesRat chondrocytes cultured in 6-well plates were pretreated first with Aucubin and then with Aucubin (1,10,20,50 μM) and IL-1β for 24 h. Western blot were used to identified the expression of apoptosis-related protein Bcl-2, Bax, Cleaved Caspase-3 and Cleaved Caspase-9.Statistical analysisAll experiments were performed in triplicate and the results are provided as mean values from three independent experiments. Results are presented as mean ±SD. All analyses were performed using SPSS version 16.0 software. For multiple comparisons, one-way analysis of variance (ANOVA) was used depending on the experiment design and the Dunnett method or Dunnett’s T3 method was used. Statistical differences between nonparametric for different groups were compared using K Independent Samples Test procedure. A p value of <0.05 was considered statistically significant.Results:1. Aucubin have no cytotoxic effects on chondrocytesAucubin at the concentrations we used in the present study did not decrease the viabilities of chondrocytes significantly as compared with controls at 24 h and 48 h (F24h=1.342, P24h=0.320; F48h=0.103, P48h=0.979, Table 1-1), indicating that Aucubin at the concentration range of 1-50 μM did not have cytotoxic effects on chondrocytes at either 24 h or 48 h.2. Inhibition effects of Aucubin on IL-1β-Induced inflammatory and Catabolic activities in ChondrocytesPCR results illustrated that relative gene expressions of MMP-3, MMP-9, MMP-13, iNOS, and COX-2 significantly be up-regulated after stimulation with IL-1β (Fig.2). Aucubin remarkably inhibited the gene expression of MMP-3, MMP-9, MMP-13, iNOS and COX-2 in a dose-dependent manner in contrast with chondrocytes stimulated with IL-1β alone at 24 h (FMMP-3=28.045, P<0.05; FMMP-9=19.569, P<0.001; FMMP-13=9.679, P=0.002; FiNOS=16.181, P<0.001; F COX-2=39.690, P<0.001 Table 1-2,1-3,1-4,1-5,1-6,1-7).Western blot analysis showed that protein expression of MMP-3, MMP-9, MMP-13, iNOS and COX-2 increased in IL-1β treated chondrocytes compared to untreated chondrocytes, and Aucubin remarkably inhibited their elevated expression in a dose-dependent manner at 24 h time point (Fig.3). These were consistent with results of PCR, indicating that Aucubin may have an anti-inflammatory and anti-catabolic effect on IL-1β-stimulated chondrocytes.3. Inhibition effects of Aucubin on IL-1β-Induced activation of NF-κB in ChondrocytesWestern blot analysis at the 4 time points showed that IL-1β markedly induced the phosphorylation of IKKα/β, IκBα and NF-κB p65 in a time-dependent manner, with the phosphorylation response reaching the peak at approximately 2 h and decreasing rapidly at 6 h. The Western blotting at 2 h showed that IL-1β markedly induced the phosphorylation of IKKa/p, IκBα and NF-κB p65, and that the phosphorylation was significantly reversed by Aucubin in a dose-dependent manner.Immunofluorescence microscopy was employed to reveal translocation of p65 subunit of NF-κB from the cytoplasm to the nucleus in chondrocytes in response to NF-κB activation by IL-1β. Chondrocytes remained either unstimulated or with 10 ng/ml IL-1β alone for 2 h or were co-treated with 10 ng/ml IL-1β and 50 μM Aucubin for 2 h before indirect immunolabelling with anti-p65 antibody. FITC-coupled secondary antibodies were used. All results from western blot and Immunofluorescence microscopy indicated that IL-1β-Induced activation of NF-κB can be blocked by Aucubin.4. Inhibition effects of Aucubin on IL-1β-Induced ROS generation in ChondrocytesConfocal Laser Scanning Microscope were used to identified the ROS level, IL-1β challenged group in chondrocytes significant increase of ROS level compared to both unmodified control cells. When chondrocytes were incubated with Aucubin at concentration of 50μM, it showed a decrease in ROS generation in comparison with IL-1β challenged group(F=6.003, P=0.005, Table 2-2).5. Inhibition effects of Aucubin on IL-1β-Induced apoptosis in ChondrocytesResults from FCM analysis demonstrated that IL-1β induce apoptosis of chondrocytes. When chondrocytes were incubated with Aucubin at concentration of 1μM, 10μM,20μM,50μM and IL-1β, it showed a decrease in percentage of apoptosis on chondrocytes in comparison with IL-1β challenged group, in a dose-dependent manner (F=86.66, P<0.001, Table 2-1).6. Inhibition effects of A ucubin on IL-1β-Induced apoptosis-related proteinResults from Western blot demonstrated that IL-1β induced apoptosis of chondrocytes and expression of pro-apoptosis protein is up-regulated and expression of anti-apoptosis protein is down-regulated. The pro-apoptosis protein level of Bax, Cleaved Caspase-3 and Cleaved Caspase-9 is promoted by stimulation of IL-1β. In Aucubin and IL-1β group, Bax, Cleaved Caspase-3 and Cleaved Caspase-9 protein expression levels is decreased.while the Bcl-2, the anti-apoptosis protein, is not increased after IL-1β stimulation, in contrast, is decreased. In Aucubin and IL-1β group, expression of Bcl-2 is elevated.Conclusions:1. Aucubin exhibited an ability to antagonize the IL-1β-dependent up-regulation of MMP-3, MMP-9, MMP-13, COX-2, iNOS expression and the production of NO, indicating the anti-inflammatory and chondro-protective effect of Aucubin in IL-1β-challenged rat chondrocytes.2. Treatment of Aucubin suppressed IL-1β-induced phosphorylation and nuclear translocation of NF-κB in the chondrocytes, revealing the molecular mechanism of Aucubin action.3. Aucubin decrease level of ROS generation on IL-1β-Induced chondrocytes.4. Aucubin inhibit IL-1β induced apoptosis of chondroncytes by decreasedexpression of pro-apoptosis protein and promoted expression of anti-apoptosis protein. |
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