Salidroside attenuates interleukin-1β-induced inflammation in human osteoarthritis chondrocytes

Salidroside, a bioactive constituent isolated from Rhodiola rosea, has been reported to have anti-inflammatory effects. However, the effects of salidroside on interleukin (IL)-1β-stimulated osteoarthritis (OA) chondrocytes remain to be elucidated. Thus, this study aimed to evaluate the anti-inflammatory effects of salidroside on IL-1β-stimulated human OA chondrocytes and explore its underlying mechanisms. Our results showed that salidroside significantly inhibited the production of nitric oxide and prostaglandin E-2, as well as suppressed the expression of inducible nitric oxide synthase and cyclooxygenase-2 in IL-1β-stimulated chondrocytes ( P < .05). In addition, salidroside also suppressed IL-1β-induced matrix metalloproteinases production in human OA chondrocytes ( P < .05). Furthermore, pretreatment with salidroside prevented IL-1β-induced NF-κB activation in OA chondrocytes ( P < .05). In conclusion, the current study demonstrated that salidroside inhibited the IL-1β-induced inflammatory response in OA chondrocytes via inhibition of NF-κB activation.     

DNA methylation at promoter regions of interleukin 1B, interleukin 6, and interleukin 8 in non-small cell lung cancer

Epidemiologic and experimental evidences support the concept that inflammation promotes the development and progression of cancers. Interleukins (ILs) regulate the expression of several molecules and signaling pathways involved in inflammation. High expression of some ILs in the tumor microenvironment has been associated with a more virulent tumor phenotype. To examine the role of IL-1β, IL-6, and IL-8 in non-small cell lung cancer, we measured mRNA levels and promoter DNA methylation in a panel of cultured human lung cells (n = 23) and in matched pair lung tumor versus adjacent non-tumorous tissues (n = 24). We found that lung cancer cells or tissues had significantly different DNA methylation and mRNA levels than normal human bronchial epithelial cells or adjacent non-tumorous tissues, respectively. High DNA methylation of ILs promoters in lung cancer cells or tissues was associated with low mRNA levels. We found an inverse correlation between DNA methylation of IL1B, IL6, and IL8 gene promoters and their corresponding mRNA levels, such inverse correlation was more significant for IL1B (i.e., all cancer cell lines used in this study had a hypermethylated IL1B promoter which was associated with silencing of the gene). Our results underline for the first time the role of epigenetic modifications in the regulation of the expression of key cytokines involved in the inflammatory response during lung cancer development.     

Caesalpinia sappan extract inhibits IL1β-mediated overexpression of matrix metalloproteinases in human chondrocytes

Exacerbated production of matrix metalloproteinases (MMPs) is a key event in the progression of osteoarthritis (OA) and represents a promising target for the management of OA with nutraceuticals. In this study, we sought to determine the MMP-inhibitory activity of an ethanolic Caesalpinia sappan extract (CSE) in human OA chondrocytes. Thus, human articular chondrocytes isolated from OA cartilage and SW1353 chondrocytes were stimulated with Interleukin-1beta (IL1β), without or with pretreatment with CSE. Following viability assays, the production of MMP-2 and MMP-13 was assessed using ELISA, whereas mRNA levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13 and TIMP-1, TIMP-2, TIMP-3 were quantified using RT-qPCR assays. Chondrocytes were co-transfected with a MMP-13 luciferase reporter construct and NF-kB p50 and p65 expression vectors in the presence or absence of CSE. In addition, the direct effect of CSE on the proteolytic activities of MMP-2 was evaluated using gelatin zymography. We found that CSE significantly suppressed IL1β-mediated upregulation of MMP-13 mRNA and protein levels via abrogation of the NF-kB(p65/p50)-driven MMP-13 promoter activation. We further observed that the levels of IL1β-induced MMP-1, MMP-3, MMP-7, and MMP-9 mRNA, but not TIMP mRNA levels, were down-regulated in chondrocytes in response to CSE. Zymographic results suggested that CSE did not directly interfere with the proteolytic activity of MMP-2. In summary, this study provides evidence for the MMP-inhibitory potential of CSE or CSE-derived compounds in human OA chondrocytes. The data indicate that the mechanism of this inhibition might, at least in part, involve targeting of NF-kB-mediated promoter activation.     

Cyclooxygenase-2 activity is necessary for the angiogenic properties of oncostatin M.

Macrophages play a major role in angiogenesis. We recently reported that oncostatin M (OSM), a cytokine of the interleukin (IL)-6 family secreted by macrophages, has a potent angiogenic activity on human microvascular endothelial cells (HMEC-1), but has no effect on macrovascular cells (human umbilical vein endothelial cells (HUVECs)). In this work, we show that in HMEC-1, OSM (0.5-2.5 ng/ml), leukemia inhibitory factor (LIF) (25 ng/ml), bFGF (25 ng/ml) and IL-1beta (5 ng/ml) induced production of cyclooxygenase (COX)-2. In contrast, in HUVECs, neither OSM nor LIF induced COX-2 mRNA, suggesting that COX-2 might be implicated in the angiogenic activity of OSM. This was confirmed by the inhibiting effect on OSM-induced HMEC-1 proliferation of specific COX-2 inhibitors. In vivo studies confirmed this findings. We conclude that induction of COX-2 by OSM is necessary for its angiogenic activity, but is not sufficient since IL-1beta, which also induces COX-2 in HMEC-1, has only a poor proliferative effect.     

Inhibition of NF‐κB is required for oleanolic acid to downregulate PD‐L1 by promoting DNA demethylation in gastric cancer cells

Gastric cancer is one of the most common causes of cancer‐related death worldwide. Immunotherapy via programmed cell death protein 1 (PD‐1)/programmed cell death‐ligand 1 (PD‐L1) blockade has shown benefits for gastric cancer. Epigenetic DNA methylation critically regulates cancer immune checkpoints. We investigated how the natural compound oleanolic acid (OA) affected PD‐L1 expression in gastric cancer cells. Interleukin‐1β (IL‐1β) at 20 ng/mL was used to stimulate human gastric cancer MKN‐45 cells. IL‐1β significantly increased PD‐L1 expression, which was abolished by OA. Next, OA‐treated MKN‐45 cells were co‐cultured with activated and PD‐1‐overexpressing Jurkat T cells. OA restored IL‐2 levels in the co‐culture system and increased T cell killing toward MKN‐45 cells. Overexpression of PD‐L1 eliminated OA‐enhanced T cell killing capacity; however, PD‐1 blocking antibody abrogated the cytotoxicity of T cells. Moreover, OA abolished IL‐1β‐increased DNA demethylase activity in MKN‐45 cells. DNA methyltransferase inhibitor 5‐azacytidine rescued OA‐reduced PD‐L1 expression; whereas DNA demethylation inhibitor gemcitabine inhibited PD‐L1 expression, and, in combination with OA, provided more potent inhibitory effects. Furthermore, OA selectively reduced the expression of DNA demethylase TET3 in IL‐1β‐treated MKN‐45 cells, and overexpression of TET3 restored OA‐reduced PD‐L1 expression. Finally, OA disrupted nuclear factor κB (NF‐κB) signaling IL‐1β‐treated MKN‐45 cells, and overexpression of NF‐κB restored OA downregulation of TET3 and PD‐L1. The cytotoxicity of T cells toward MKN‐45 cells was also weakened by NF‐κB overexpression. Altogether, OA blocked the IL‐1β/NF‐κB/TET3 axis in gastric cancer cells, leading to DNA hypomethylation and downregulation of PD‐L1. Our discoveries suggested OA as an epigenetic modulator for immunotherapy or an adjuvant therapy against gastric cancer.     

CYTOKINES INVOLVING gp130 IN SIGNAL TRANSDUCTION SUPPRESSED GROWTH OF A MOUSE HYBRIDOMA CELL LINE AND ENHANCED ITS ANTIBODY PRODUCTION

Three cytokines, interleukin 6 (IL-6), leukaemia inhibitory factor (LIF), and oncostatin M (OSM), that bind to composite receptors including a common signal transducer gp130 suppressed proliferation of a mouse B-cell hybridoma cell line 2E3-O cultured in serum-free medium, while they enhanced antibody production of the cells. The specific growth rate of the cells reduced from 1.0/day for control to 0.6/day for the cultures supplemented with IL-6, LIF, or OSM at 1, 4, or 2 ng/ml, respectively. The antibody productivity increased five-fold when the cells were cultured with IL-6, LIF, or OSM at 1, 25, or 20 ng/ml, respectively. Transforming growth factor β1 (TGF-β1) similarly suppressed growth of the cells at the concentration of 5 ng/ml, while it did not enhance the antibody production. Cell cycle analysis revealed that IL-6 induced the cells to be arrested at G₁phase of the cell cycle more intensively than TGF-β1, indicating that IL-6 and TGF-β1 suppressed the growth through mutually different mechanisms. As a whole, this work suggests that gp130, which is commonly involved in each receptor for IL-6, LIF, OSM, transduces signals for suppressing proliferation and possibly for enhancing antibody production in the hybridoma cells.     

Augmentation of ADAMTS9 gene expression by IL‐1β is reversed by NFκB and MAPK inhibitors,but not PI3 kinase inhibitors

The pathways involved in the regulation of a disintegrin and metalloproteinase with thrombospondin motifs 9 (ADAMTS9) expression have not yet been elucidated. Therefore, the aim of this study was to investigate the involvement of nuclear factor‐κB (NF‐κB), mitogen activated protein kinases (MAPK) and Phosphatidylinositol 3‐kinase (PI3 kinase) in ADAMTS9 gene regulation, with special focus on the involvement of NF‐κB in IL‐1β‐induced ADAMTS9 expression. The OUMS‐27 chondrosarcoma cells were exposed to IL‐1β. They were pretreated with 20 μM PD98059 (specific inhibitor of p44/42 kinase), 10 μM SB203580 (specific inhibitor of p38 kinase), 20 μM SB600125 (MAPK inhibitor), and 1 μM Wortmannin and 10 μM LY294002 (specific inhibitors of PI3 kinase) for 30 min and subsequently incubated with IL‐1β. For the effects of NF‐κB and IκB inhibitors, cells were pretreated with curcumin or BAY117085 for 30 min and subsequently incubated with IL‐1β. BAY117085 and different concentrations of curcumin were applied to the cells just after the first experiment to determine their concentration effect on ADAMTS9 gene expression. After total RNA was extracted, they were reversely transcribed with random primers and then real‐time polymerase chain reaction (PCR) was performed on cDNA samples. There was a significant difference between control and stimulated cells in terms of ADAMTS9/β‐actin ratio. Wortmannin and LY294002 did not have any repressive effect on the OUMS‐27 whereas SB203580 and SP600125 were found to decrease the expression of ADAMTS9 gene. BAY 117085 and curcumin, which are two NF‐κB inhibitors, led to a decrease in the ratio of ADAMTS9/β‐actin. As a conclusion, the pathways MAPK and NF‐κB were thought to be responsible pathways for the induction of ADAMTS9 gene. Copyright © 2012 John Wiley & Sons, Ltd.     

Schisandra extract ameliorates arthritis pathogenesis by suppressing the NF-κB and MAPK signalling pathways

Schisandra chinensis is a medicinal plant used to treat various diseases. Extracts from the leaves or fruits of S. chinensis and their components are used in osteoarthritis (OA). The OA inhibitory effect of schisandrol A, one of its components, has been previously confirmed. We aimed to confirm the OA inhibitory effect of Schisandra (including components like schisandrol A) to identify why the inhibitory effect of the Schisandra extract is better. First, we investigated the effects of the Schisandra extract on OA as a potential therapeutic. Experimental OA was induced in a mouse model via destabilized medial meniscus surgery. The animals were orally administered the Schisandra extract; the inhibition of cartilage destruction was confirmed using histological analysis. In vitro analysis showed that the Schisandra extract attenuated osteoarthritic cartilage destruction by regulating IL-1β-induced MMP3 and COX-2 levels. The Schisandra extract inhibited IL-1β-induced degradation of IκB (NF-κB pathway) and IL-1β-induced phosphorylation of p38 and JNK (mitogen-activated protein kinase (MAPK) pathway). RNA-sequencing analysis showed that the Schisandra extract decreased the expression of IL-1β-induced MAPK and NF-κB signalling pathway-related genes more than schisandrol A alone. Therefore, Schisandra extract may be more effective than schisandrol A in preventing OA progression by regulating MAPK and NF-κB signalling.     

Aberrant methylation of the IL‐12B promotor region contributes to the risk of developing ovarian endometriosis

Studies have shown that aberrant expression of IL‐12p40, which is encoded by the interleukin‐12B (IL‐12B) gene, may be involved in the development of endometriosis. In this study, we investigated the role of aberrant methylation of the IL‐12B promoter region and its associated expression in the development of ovarian endometriosis. By using pyrosequencing, we analyzed the methylation level of the IL‐12B promoter region in eutopic and ectopic endometrium of patients with ovarian endometriosis and normal endometrium of control women. The expression of IL‐12B mRNA was detected by quantitative real‐time PCR. The results showed that the methylation level of the IL‐12B promoter region in ectopic and eutopic endometrium of patients with ovarian endometriosis was significantly lower than that in endometrium of women without endometriosis ( p < 0.001 and p = 0.041, respectively). In contrast, mRNA levels were significantly increased in ectopic and eutopic endometrium of patients with ovarian endometriosis compared to those in endometrium of women without endometriosis ( p < 0.001 and p = 0.042, respectively). Correlation analysis showed that the methylation level of the IL‐12B promoter region was negatively correlated with mRNA levels of IL‐12B ( p < 0.001). Our data suggested that aberrant methylation of the IL‐12B promoter region may be responsible for aberrant IL‐12B mRNA expression in endometrium tissue of women, which may be associated with the development of ovarian endometriosis in northern Chinese women.     

Anemonin attenuates osteoarthritis progression through inhibiting the activation of IL‐1β/NF‐κB pathway

The osteoarthritis (OA) progression is now considered to be related to inflammation. Anemonin (ANE) is a small natural molecule extracted from various kinds of Chinese traditional herbs and has been shown to inhibiting inflammation response. In this study, we examined whether ANE could attenuate the progression of OA via suppression of IL‐1β/NF‐κB pathway activation. Destabilization of the medial meniscus (DMM) was performed in 10‐week‐old male C57BL/6J mice. ANE was then intra‐articularly injected into joint capsule for 8 and 12 weeks. Human articular chondrocytes and cartilage explants challenged with interleukin‐1β (IL‐1β) were treated with ANE. We found that ANE delayed articular cartilage degeneration in vitro and in vivo. In particular, proteoglycan loss and chondrocyte hypertrophy were significantly decreased in ANE ‐treated mice compared with vehicle‐treated mice. ANE decreased the expressions of matrix metalloproteinase‐13 (MMP13), A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), collagen X (Col X) while increasing Aggrecan level in murine with DMM surgery. ANE treatment also attenuated proteoglycan loss in human cartilage explants treated with IL‐1β ex vivo. ANE is a potent protective molecule for OA; it delays OA progression by suppressing ECM loss and chondrocyte hypertrophy partially by suppressing IL‐1β/NF‐κB pathway activation.     

Protective effects of calcitonin on IL-1 stimulated chondrocytes by regulating MMPs/TIMP-1 ratio via suppression of p50-NF-κB pathway

The aim of this study was to investigate the effects and underlying mechanisms of calcitonin (CT) on interleukin 1 beta (IL-1β) stimulated human chondrocytes. IL-1β (5 ng/mL) was added into chondrocytes to establish osteoarthritis (OA) model in vitro. Different concentrations of CT (0.1, 0.5, 1, 5, 10 and 50 nM) were used for treating IL-1β stimulated chondrocytes. Cell viability of chondrocytes was measured by cell counting kit-8 (CCK8) method. Western blotting was performed to evaluate the expression of matrix metalloproteinases (MMP-13), tissue inhibitor of metalloproteinases 1 (TIMP-1), p50 and p38. CT inhibited MMP-13 expression and promoted TIMP-1 expression in the IL-1β stimulated human chondrocytes. The CT-mediated alteration of MMP-13/TIMP-1 ratio was partially attributed to the inactivation of the p50- nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway by suppressing p50 in IL-1β stimulated chondrocytes. CT might play a protective role in IL-1β stimulated OA model via p50-NF-κB pathway.

Abbreviations: CT: calcitonin; IL-1β: interleukin-1β; MMP-13: matrix metalloproteinases-13; TIMP-1: tissue inhibitors of metalloproteinases-1.     

White blood cell global methylation and IL-6 promoter methylation in association with diet and lifestyle risk factors in a cancer-free population

Altered levels of global DNA methylation and gene silencing through methylation of promoter regions can impact cancer risk, but little is known about their environmental determinants. We examined the association between lifestyle factors and levels of global genomic methylation and IL-6 promoter methylation in white blood cell DNA of 165 cancer-free subjects, 18–78 years old, enrolled in the COMIR (Commuting Mode and Inflammatory Response) study, New York, 2009–2010. Besides self-administrated questionnaires on diet and physical activity, we measured weight and height, white blood cell (WBC) counts, plasma levels of high sensitivity C-reactive protein (hs-CRP), and genomic (LINE-1) and gene-specific methylation (IL-6) by pyrosequencing in peripheral blood WBC. Mean levels of LINE-1 and IL-6 promoter methylation were 78.2% and 57.1%, respectively. In multivariate linear regression models adjusting for age, gender, race/ethnicity, body mass index, diet, physical activity, WBC counts and CRP, only dietary folate intake from fortified foods was positively associated with LINE-1 methylation. Levels of IL-6 promoter methylation were not significantly correlated with age, gender, race/ethnicity, body mass index, physical activity or diet, including overall dietary patterns and individual food groups and nutrients. There were no apparent associations between levels of methylation and inflammation markers such as WBC counts and hs-CRP. Overall, among several lifestyle factors examined in association with DNA methylation, only dietary folate intake from fortification was associated with LINE-1 methylation. The long-term consequence of folate fortification on DNA methylation needs to be further evaluated in longitudinal settings.     

Global and gene-specific promoter methylation analysis in primary hyperparathyroidism

Epigenetic mechanisms involved in primary hyperparathyroidism are poorly understood as studies are limited. In order to understand the role of aberrant DNA promoter methylation in the pathogenesis of parathyroid tumors, we have quantified the CpG island promoter methylation density of several candidate genes including APC (promoter 1A and 1B), β-catenin (CTNNB1), CASR, CDC73/HRPT2, MEN1, P16 (CDKN2A), PAX1, RASSF1A, SFRP1 and VDR in 72 parathyroid tumors and 3 normal parathyroid references using bisulfite pyrosequencing. Global methylation levels were assessed for LINE-1. We also compared methylation levels with gene expression levels measured by qRT-PCR for genes showing frequent hypermethylation. The adenomas displayed frequent hypermethylation of APC 1A (37/66; 56%), RASSF1A (34/66; 52%) and β-catenin (19/66; 29%). One of the three atypical adenomas was hypermethylated for APC 1A. The three carcinomas were hypermethylated for RASSF1A and SFRP1, and the latter was only observed in this subtype. The global methylation density was similar in tumors (mean 70%) and parathyroid reference samples (mean 70%). In general, hypermethylated genes had reduced expression in the parathyroid adenomas using qRT-PCR. Among the adenomas, methylation of APC 1A correlated with adenoma weight (r = 0.306, p < 0.05). Furthermore, the methylation status of RASSF1A correlated with each of APC 1A (r = 0.289, p < 0.05) and β-catenin (r = 0.315, p < 0.01). Our findings suggest a role for aberrant DNA promoter methylation of APC 1A, β-catenin and RASSF1A in a subset of parathyroid tumors.     

Detection of Polygalacturonase in Kiwifruit during Ripening

Oligosaccharides produced during the course of the hydrolysis of 25% N-acetylated chitosan by Streptomyces griseus chitinase were fractionated by CM-Sephadex C-25 and Toyopearl HW-40F column chromatographies. Sugar compositions and sequences of main oligosaccharides were identified by N-acetylation, exo-splitting with β-GlcNAcase and β-GlcNase, and nitrous acid degradation. In addition to N-acetylated saccharides, GlcNAc, (GlcNAc)₂, and (GlcNAc)₃, hetero-chitooligosaccharides such as GlcN · GlcNAc, GlcN · GlcNAc · GlcNAc, GlcN · GlcN · GlcNAc, GlcN · GlcNAc · GlcNAc · GlcNAc, GlcNAc · GlcN · GlcNAc · GlcNAc, GlcN · GlcNAc · GlcN · GlcNAc, and GlcN · GlcN · GlcNAc · GlcNAc were identified. These results indicate that Streptomyces griseus chitinase specifically cleaves the N-acetyl-β-d-glucosaminidic linkages in partially N-acetylated chitosan.     

Cytokine‐induced interleukin‐1 receptor antagonist protein expression in genetically engineered equine mesenchymal stem cells for osteoarthritis treatment

Background

A combination of tissue engineering methods employing mesenchymal stem cells (MSCs) together with gene transfer takes advantage of innovative strategies and highlights a new approach for targeting osteoarthritis (OA) and other cartilage defects. Furthermore, the development of systems allowing tunable transgene expression as regulated by natural disease‐induced substances is highly desirable.

Methods

Bone marrow‐derived equine MSCs were transduced with a lentiviral vector expressing interleukin‐1 receptor antagonist (IL‐1Ra) gene under the control of an inducible nuclear factor‐kappa B‐responsive promoter and IL‐1Ra production upon pro‐inflammatory cytokine stimulation [tumor necrosis factor (TNF)α, interleukin (IL)‐1β] was analysed. To assess the biological activity of the IL‐1Ra protein that was produced and the therapeutic effect of IL‐1Ra‐expressing MSCs (MSC/IL‐1Ra), cytokine‐based two‐ and three‐dimensional in vitro models of osteoarthritis using equine chondrocytes were established and quantitative real‐time polymerase chain reaction (PCR) analysis was used to measure the gene expression of aggrecan, collagen IIA1, interleukin‐1β, interleukin‐6, interleukin‐8, matrix metalloproteinase‐1 and matrix metalloproteinase‐13.

Results

A dose‐dependent increase in IL‐1Ra expression was found in MSC/IL‐1Ra cells upon TNFα administration, whereas stimulation using IL‐1β did not lead to IL‐1Ra production above the basal level observed in nonstimulated cells as a result of the existing feedback loop. Repeated cycles of induction allowed on/off modulation of transgene expression. In vitro analyses revealed that IL‐1Ra protein present in the conditioned medium from MSC/IL‐1Ra cells blocks OA onset in cytokine‐treated equine chondrocytes and co‐cultivation of MSC/IL‐1Ra cells with osteoarthritic spheroids alleviates the severity of the osteoarthritic changes.

Conclusions

Thus, pro‐inflammatory cytokine induced IL‐1Ra protein expression from genetically modified MSCs might represent a promising strategy for osteoarthritis treatment.     

Effects of different omega-3 sources,fish oil,krill oil,and green-lipped mussel against cytokine-mediated canine cartilage degradation

Our purpose was to evaluate the protective effect of three marine omega-3 sources, fish oil (FO), krill oil (KO), and green-lipped mussel (GLM) against cartilage degradation. Canine cartilage explants were stimulated with either 10 ng/mL interleukin-1β (IL-1β) or IL-1β/oncostatin M (10 ng/mL each) and then treated with various concentrations of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA; 3 and 30 μg/mL), FO, KO, or GLM (250, 500, and 1000 μg/mL) for 28 days. Gene expression was then investigated in primary canine chondrocytes. Our results showed that DHA and EPA as well as omega-3 sources could suppress matrix degradation in cytokine-induced cartilage explants by significantly reducing the increase of sulfated glycosaminoglycans (s-GAGs) and preserving uronic acid and hydroxyproline content (except GLM). These agents were not able to reduce IL-1β-induced IL1B and TNFA expression but were able to down-regulate the expression of the catabolic genes MMP1, MMP3, and MMP13 and up-regulate the anabolic genes AGG and COL2A1; FO and KO were especially effective. Our findings indicated that FO and KO were superior to GLM for their protective effect against proteoglycan and collagen degradation. Hence, FO and KO could serve as promising sources of chondroprotective agents.