Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Diacerein inhibits the pro-atherogenic & pro-inflammatory effects of IL-1 on human keratinocytes & endothelial cells

  • Girish C. Mohan ,

    Contributed equally to this work with: Girish C. Mohan, Huayi Zhang

    Affiliation Department of Dermatology, University of Illinois at Chicago (UIC)—College of Medicine, Chicago, IL, United States of America

    ⨯
  • Huayi Zhang ,

    Contributed equally to this work with: Girish C. Mohan, Huayi Zhang

    Affiliation Department of Dermatology, University of Illinois at Chicago (UIC)—College of Medicine, Chicago, IL, United States of America

    ⨯
  • Lei Bao,

    Affiliation Department of Dermatology, University of Illinois at Chicago (UIC)—College of Medicine, Chicago, IL, United States of America

    ⨯
  • Benjamin Many,

    Affiliation Department of Dermatology, University of Illinois at Chicago (UIC)—College of Medicine, Chicago, IL, United States of America

    ⨯
  • Lawrence S. Chan

    larrycha@uic.edu

    Affiliations Department of Dermatology, University of Illinois at Chicago (UIC)—College of Medicine, Chicago, IL, United States of America, Department of Medicine, Jesse Brown Veterans Affairs Hospital, Chicago, IL, United States of America, Department of Medicine, Capt. James A. Lovell FHCC, North Chicago, IL, United States of America

    ⨯

Abstract

We investigated IL-1-induced regulation of genes related to inflammation and atherogenesis in human keratinocytes and endothelial cells, and if ‘diacerein’, an oral IL-1 inhibiting drug currently approved for use in osteoarthritis, would reverse IL-1’s effects on these cells. Primary human keratinocytes and coronary artery endothelial cells were treated with either IL-1α or IL-1β, with and without diacerein. Using PCR-array, we assessed differential gene-expression regulated by IL-1 and diacerein. We identified 34 pro-atherogenic genes in endothelial cells and 68 pro-inflammatory genes in keratinocytes significantly (p<0.05) regulated at least 2-fold by IL-1, in comparison to control. Diacerein completely or partially reversed this regulation on almost all genes. Using ELISA, we confirmed diacerein’s ability to reverse IL-1-driven gene-regulation of 11 selected factors, at the protein level. The results support a novel idea that diacerein acts as an inhibitor of the pro-atherogenic and pro-inflammatory effects of IL-1. Diacerein may have therapeutic applications to diminish IL-1-induced skin inflammation in psoriasis and attenuate IL-1-induced development of atherosclerosis. Further investigation into diacerein’s effect on skin inflammation, atherogenesis and cardiovascular risk in animal models or humans is warranted.

Citation: Mohan GC, Zhang H, Bao L, Many B, Chan LS (2017) Diacerein inhibits the pro-atherogenic & pro-inflammatory effects of IL-1 on human keratinocytes & endothelial cells. PLoS ONE 12(3): e0173981. https://doi.org/10.1371/journal.pone.0173981

Editor: Andrea Caporali, University of Edinburgh, UNITED KINGDOM

Received: March 23, 2015; Accepted: March 1, 2017; Published: March 21, 2017

Copyright: © 2017 Mohan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by Orville J. Stone Endowed Professorship (L.S. Chan); Albert H. and Mary-Jane Slepyan Endowed Fellowship (L. Bao); University of Illinois Tanja Andric Memorial Endowment Fund (G.C. Mohan).

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: IL-1α, interleukin-1alpha; IL-1β, interleukin-1beta; EC, primary human coronary artery endothelial cell; KC, primary human keratinocyte

Introduction

Psoriasis, a chronic inflammatory skin disease, affects up to 8.5% of adult populations [1]. Psoriasis was previously considered a solely cutaneous entity but recent studies, including a meta-analysis, have shown that moderate-to-severe psoriasis patients have increased cardiovascular risk, with more frequent atherosclerotic events (ex. myocardial infarction) and life expectancy reduction by up to 4 years [2–4]. Although one study showed no link between psoriasis and cardiovascular morbidity [5], data in the literature predominantly demonstrates that an association exists.

Interleukin-1alpha (IL-1α) [6, 7] and interleukin-1beta (IL-1β) [8], the two major subunits of IL-1, are implicated in psoriasis pathogenesis. Increased expression of IL-1α and IL-1β have been found in psoriatic lesional skin in mouse models and in human subjects, as these cytokines directly contribute to the inflammation present in the skin [6, 8, 9]. However, inflammation in psoriasis is not confined to skin; evidence of chronic systemic inflammation exists [10]. The systemic inflammatory milieu in psoriasis likely contributes to the atherosclerosis (considered an inflammatory disease) present in this disease [11]. Specifically, increased levels of IL-1 have been found in this systemic inflammatory milieu [12]. Since IL-1α and IL-1β have been shown to contribute to vascular inflammation and atherosclerosis [13–15], the increased levels of serum IL-1 noted in psoriasis may be responsible for promoting atherosclerosis.

Diacerein is an IL-1 inhibitor, approved in Europe as an oral anti-inflammatory treatment for osteoarthritis. The aim of this study is to investigate IL-1-driven regulation of genes related to inflammation and atherogenesis in human coronary artery endothelial cells (EC) and keratinocytes (KC), and to study the effects of diacerein on this gene regulation. ECs and KCs were utilized because they are directly involved in atherogenesis and psoriasis pathogenesis, respectively [8, 16].

Results

In endothelial cells treated with IL-1α

PCR-Array: mRNA expression of 16 of 84 atherosclerosis-related genes were significantly regulated at least 2-fold by IL-1α alone in comparison to control (p<.05), ranging from 8.3-fold (SELPLG) to 108.4-fold (TGFB2) up-regulation. Other genes regulated include ACE, BCL2L1, CSF2, FAS, ICAM1, LIF, MMP1, NR1H3, PLIN2, PPARG, SELE, ITGA2, TGFB1 and TNFAIP3. Adding 15µM diacerein partially or completely reversed these regulations in 15 of the 16 genes (Table 1). Real-time PCR verified the PCR-Array results of IL-1-induced regulation and their reversal by diacerein for ICAM1 and SELE (these and other real-time PCR data not shown).

thumbnail
Download:
Table 1. Atherosclerosis PCR-array: genes regulated by IL-1α and diacerein in ECs1

https://doi.org/10.1371/journal.pone.0173981.t001

ELISA: quantitative protein expression of ACE, SELE and TGFB2 confirmed their up-regulation found on the mRNA level. Diacerein reversed IL-1α-induced protein up-regulation of these 3 genes. (Fig 1)

thumbnail
Download:
Fig 1. Regulation of genes at protein levels with IL-1α and diacerein in ECs.

Protein expression by ELISA of TGF-B2, ACE, and SELE. Human endothelial cells were treated for 24 hours with IL-1α and diacerein as described in Methods. Cell culture supernatant was measured by ELISA assays according to the manufacturer’s instructions. Values are expressed as the mean ± SEM (n = 4). *P < 0.05 vs. control.

https://doi.org/10.1371/journal.pone.0173981.g001

In endothelial cells treated with IL-1β

PCR-Array: mRNA expression of 18 of 84 atherosclerosis-related genes were significantly regulated at least 2-fold by IL-1β alone in comparison to control (p<.05), ranging from 2.3-fold (LDLR) to 1378.9-fold (CSF2) up-regulation. Other genes regulated include BCL2A1, BIRC3, CCL2, CCL5, CD44, CSF1, ICAM1, IL1A, NFKB1, SELE, SELL, SERPINB2, TNC, TNF, TNFAIP3 and VCAM1. Adding 50µM diacerein partially or completely reversed these regulations in all 18 genes (Table 2)

thumbnail
Download:
Table 2. Atherosclerosis PCR-array: genes regulated by IL-1β and diacerein in ECs1

https://doi.org/10.1371/journal.pone.0173981.t002

ELISA: quantitative protein expression of SELE, VCAM1, CSF2, TNF and CCL5 confirmed their up-regulation found on the mRNA level. Diacerein reversed IL-1β-induced protein up-regulation of these 5 genes. (Fig 2)

thumbnail
Download:
Fig 2. Regulation of genes at the protein level with IL-1β and diacerein in ECs.

Protein expression by ELISA of SELE, VCAM1, CSF2, TNF-alpha, and CCL5. Human primary keratinocytes were treated for 24 hours with IL-1β and diacerein as described in Methods. Cell culture supernatant was measured by ELISA assays according to the manufacturer’s instructions. Values are expressed as the mean ± SEM (n = 4). *P < 0.05 vs. control.

https://doi.org/10.1371/journal.pone.0173981.g002

In keratinocytes treated with IL-1α

PCR-Array: mRNA expression of 24 of 370 inflammation-related genes were significantly regulated at least 2-fold by IL-1α alone in comparison to control (p<.05), ranging from 2.1-fold (CCL20) to 377.2-fold (IL-9) up-regulation. Other genes regulated include C3, CCL20, CSF2, CSF3, CXCL2, CXCL3, CXCL6, CXCR3, IL17C, IL17RB, IL23A, IL36G, IL9, ITGB2, NAMPT, NOX5, PTAFR, S100A8, SERPINA3, SPRED1, TNFSF10, and TNFSF18. Adding 10µM diacerein partially or completely reversed these regulations in all 24 genes (Table 3). Real-time PCR verified the PCR-Array results of IL-1-induced regulation and their reversal by diacerein for CXCL2.

thumbnail
Download:
Table 3. Inflammation PCR-array: genes regulated by IL-1α and diacerein in KCs1

https://doi.org/10.1371/journal.pone.0173981.t003

ELISA: quantitative protein expression of CXCR3, IL9 and CSF2 confirmed their up-regulation found on the mRNA level. Diacerein reversed IL-1α-induced protein regulation of these 3 genes. (Fig 3)

thumbnail
Download:
Fig 3. Regulation of genes at protein levels with IL-1α and diacerein in KCs.

Protein expression by ELISA of CXCR3, IL-9, CSF2. Human primary keratinocytes were treated for 24 hours with IL-1α and diacerein as described in Methods. Cell culture supernatant was measured by ELISA assays according to the manufacturer’s instructions. Values are expressed as the mean ± SEM (n = 4). *P < 0.05 vs. control.

https://doi.org/10.1371/journal.pone.0173981.g003

In keratinocytes treated with IL-1β

PCR-Array: mRNA expression of 44 of 370 inflammation-related genes were significantly regulated at least 2-fold by IL-1β alone in comparison to control (p<.05) ranging from 2.1-fold (HDAC9) to 70.9-fold (S100A8) up-regulation. Other genes regulated include CCL11, 20, 28, and 5, CXCL1, 2 and 6, GDF2, HDAC9, IL32, LIF, OLR1 and SYK (partial list). Adding 20µM diacerein partially or completely reversed these regulations in all 44 genes. (Table 4)

thumbnail
Download:
Table 4. Inflammation PCR-array: genes regulated by IL-1β and diacerein in KCs1

https://doi.org/10.1371/journal.pone.0173981.t004

Discussion

Our results demonstrate that IL-1 acts as a pro-atherogenic and pro-inflammatory mediator in ECs and KCs. Addition of diacerein yielded complete or partial reversal of IL-1-induced gene regulation.

Endothelial cells

Diacerein reverses IL-1α-induced regulation of atherosclerosis-related genes in ECs (Table 1).

IL-1α was found to be pro-atherogenic by regulating several genes in ECs including up-regulation of ACE, ICAM1and other genes; these regulations were reversed by diacerein, thus acting in an anti-atherogenic manner. ACE (angiotensin-1 converting enzyme) increases angiotensin-II, which elevates blood pressure. Elevated blood pressure accelerates atherogenesis [16] while angiotensin-II may independently promote vascular inflammation by increasing oxidative stress in vessel walls [17] ICAM1 (inter-cellular adhesion molecule-1) was also up-regulated by IL-1α in other studies [18, 19]. ICAM1, involved with inflammatory cell recruitment, was increased in atherosclerotic lesions [20]. Another study showed that reduced expression of cellular adhesion molecules in mice decreased atherogenicity [21]. Our IL-1α up-regulation of MMP1 (matrix metalloproteinase-1), was consistent with the findings of Hanemaaijer et al. [22] and was reversed by diacerein. MMP1 contributes to inflammatory cell intimal infiltration, plaque instability and rupture [23] and is correlated with increased total plaque burden [24]. Metalloproteinases are also implicated in multiple phases of atherosclerosis [25]. PLIN2 (perilipin-2), promoting foam-cell formation, was reported as a safe target for anti-atherogenic therapy [26]. SELE (E-selectin; facilitates vascular inflammatory cell infiltration) was up-regulated by IL-1α, consistent with Etter et al. [27]. We found diacerein significantly reduced EC protein expression of E-selectin, confirming functional down-regulation of the gene (Fig 1). E-selectin’s importance to atherosclerosis was demonstrated in studies showing increased E-selectin on arterial plaque surfaces [28] and reduced development of atherosclerotic lesions in mice lacking SELE [29]. Diacerein also inhibited up-regulation of SELPLG (P-selectin ligand), which helps to recruit leukocytes to the endothelium [30].

Two genes were regulated in unique ways. TNFAIP3 (TNF-alpha-induced protein-3) was down-regulated by IL-1α, in contrast to genes discussed above. However IL-1α’s effect was still pro-atherogenic because TNFAIP3 is reported to decrease inflammation and atherosclerosis in murine models [31]. Regarding LIF (leukemia inhibitory factor), two studies showed that its expression is inversely correlated with coronary atherosclerosis [32, 33]. We found IL-1α increased LIF expression, but diacerein further up-regulated the gene, enhancing LIF’s anti-atherogenic correlation.

Diacerein reverses IL-1β-induced regulation of atherosclerosis-related genes in ECs (Table 2).

IL-1β was found to be pro-atherogenic by regulating several genes in ECs including up-regulating CCL2, CCL5and other genes; these were reversed by diacerein, thus acting in an anti-atherogenic manner. CCL2 and CCL5 (chemokines) promote vascular inflammation by attracting leukocytes to vessel walls. Additionally, CCL2 is absent in endothelium in normal conditions but is increased in the setting of atherosclerosis and associated with elevated risk of myocardial infarction [34]. CD44 and VCAM1 were up-regulated by IL-1β, in accordance with another report of IL-1β up-regulation of VCAM1 in ECs [35]. Both CD44 and VCAM1 recruit inflammatory cells to the endothelium, while CD44 also interacts with hyaluronan, a glycosaminoglycan increased in atherosclerotic lesions. CD44 and VCAM1 were increased in an atherosclerosis mouse-model too [36]. Interestingly, we found IL-1β up-regulated IL-1α in ECs; IL-1α is reported to be a strong facilitator of atherogenesis and a prospective therapeutic target to reduce vascular inflammation [14]. Thus, diacerein may reverse IL-1α’s effects on atherosclerosis and also reduce its expression by ECs via suppressing IL-1β activity. TNC (tenascin-C), an extra-cellular matrix protein, is involved with induction of pro-inflammatory cytokines and metalloproteinases [37]. TNF (tumor necrosis factor-alpha), up-regulated by IL-1β in our study and another, [38] is thought to be pro-atherogenic by disrupting endothelial barrier function, inducing metalloproteinases and promoting vascular inflammation [39]. Diacerein reduced TNC and TNF protein expression by ECs (Fig 2).

Other genes regulated in ECs with unclear links to atherosclerosis.

CSF2 (granulocyte-macrophage colony-stimulating factor; recruits inflammatory cells) was down-regulated by IL-1α but highly up-regulated by IL-1β in ECs (including increased protein expression, Fig 2) and up-regulated by IL-1α in KCs. Given conflicting regulation of this gene, net effects that diacerein would exert on GM-CSF activity are unclear.

Although TGFB1 (transforming growth factor-beta1) and TGFB2 (transforming growth factor-beta2) were amplified within atherosclerotic plaques in one study [40], more convincing evidence indicates these proteins actually have an athero-protective effect [41]. Diacerein decreased TGFB1 and TGFB2 mRNA and protein expression in ECs, but only to levels that remained higher than those in untreated ECs (Fig 1). Thus, diacerein may not have great impact on atherogenesis through TGF-beta regulation.

Keratinocytes

KCs are clearly involved in psoriasis pathogenesis but unlike ECs, are not directly implicated in atherogenesis. However, gene regulation observed in KCs may still have implications for atherosclerosis in the following way. It has been suggested that the skin is a source of mediators that exert not only local inflammatory effects but also enter the circulation and cause systemic inflammation [42]; as discussed previously, systemic inflammation is associated with atherosclerosis. This may be one way skin inflammation leads to systemic and vascular inflammation, ultimately contributing to atherogenesis (Fig 4). Also, there may be a pro-inflammatory positive feedback-loop between the epidermis and endothelium which perpetuates this effect [6]. We found various inflammatory genes regulated by IL-1 in KCs, several of which may produce mediators that have atherogenic effects downstream in the vasculature. Diacerein reversed IL-1-induced regulation of all genes in KCs, thus acting in an anti-inflammatory and potentially anti-atherogenic manner.

thumbnail
Download:
Fig 4. Proposed relationship of IL-1 with keratinocytes and endothelium.

Increased levels of IL-1 locally in the skin and systemically in circulation lead to increase in pro-inflammatory and pro-atherogenic mediators in KCs and ECs. While these may have local effects in KCs leading to epidermal inflammation, they may also exert systemic effects by filtering into the circulation, ultimately leading to vascular inflammation downstream. The mediators produced by ECs promote atherogenesis as a local effect.

https://doi.org/10.1371/journal.pone.0173981.g004

Diacerein reverses IL-1α-induced regulation of genes linked to atherosclerosis in KCs (Table 3).

IL-1α is potentially pro-atherogenic by regulating several genes in KCs; these regulations were reversed by diacerein. IL17C, a cytokine typically produced by Th17 cells, is the most plentiful IL-17 isoform in psoriatic lesions [6] and may stimulate cytokine production involved in vascular inflammation [43]. IL-23A is a cytokine that helps transform naïve CD4+ T-cells into Th17 cells, which as reported, may be pro-atherogenic [44, 45]. Serum IL-23 was also significantly elevated in patients with peripheral arterial disease, a manifestation of atherosclerosis [46]. Previous reports also showed IL-1 stimulates IL-17 and IL-23 production in T-cells [47, 48] Taken together, our data shows that IL-1α may increase Th17-mediated vascular inflammation.

IL-1α also up-regulated NOX5 in KCs, whose gene-product NADPH oxidase, stimulates production of reactive oxygen species (ROS). Dysregulation of this gene is implicated in cardiovascular disease and atherosclerosis secondary to ROS-induced vascular oxidative stress [49–51]. Zhong et al. also reported that rhein (diacerein metabolite) may protect against ROS-induced EC injury [52]. IL-9 mRNA and protein expression was considerably up-regulated by IL-1α (Fig 3). In a psoriasis mouse-model, IL-9 induced Th17-related inflammatory mediators [53]. Diacerein suppression of IL-9 may be anti-atherogenic by subsequently decreasing Th17-related activity. IL-1α also up-regulated NAMPT (visfatin), a pro-inflammatory cytokine elevated in psoriasis [54]. Visfatin circulating levels are correlated with clinical manifestations of atherosclerosis [55]. TNFSF18 (GITRL) is also up-regulated in KCs. Kim et al. found increased levels of GITRL’s receptor, GITR, in atherosclerotic lesions, which when activated leads to metalloproteinase and pro-inflammatory cytokine production [56]. IL-1α-induced GITRL production by KCs may cause increased binding of GITR downstream. Diacerein also down-regulates several chemokines in KCs (CCL20, CXCL2, 3, and 6), and may oppose systemic inflammation in this way.

Diacerein reverses IL-1β regulation of genes linked to atherosclerosis in KCs (Table 4).

IL-1β is potentially pro-atherogenic by regulating several genes in KCs; these regulations were reversed by diacerein. CSF3 (granulocyte colony-stimulating factor), up-regulated by IL-1α and IL-1β, increased endothelin-1 and decreased nitric oxide synthase in an animal model, both of which are implicated in atherogenesis [57] BMP7 (bone-morphogenetic protein-7) and GDF2 (BMP9) were up-regulated by IL-1α and IL-1β, respectively. Inhibition of BMP-signaling, as diacerein provides, may impede atherosclerosis [58]. IL-1β up-regulated HDAC9; HDACs (histone deacetylases) are involved in atherogenesis and HDAC-inhibitors are a potential therapeutic target for cardiovascular disease [59]. IL-32 also is up-regulated in KCs by IL-1β; this cytokine may induce vascular inflammation and endothelial dysfunction [60]. OLR1 (Oxidized low-density lipoprotein receptor-1) is increased in atheromatous plaques, while serum levels of ORL1 are raised in coronary artery disease patients [61]. SYK (spleen tyrosine kinase) stimulates endothelin-1, implicated in atherogenesis [62]. Correspondingly, an SYK-inhibitor decreased atherogenesis in a mouse model [63]. Other genes regulated by IL-1β in KCs with potential links to atherosclerosis include CXCL1, Ebi3, IL-36G, CCL20, CCL11, PROCR, S100A8, MDK, IL20RA, CD40, IL-1β, IL36G, TLR1, TLR6 and LY75.

Diacerein reverses IL-1-induced gene regulation in KCs. By down-regulating factors from the skin that promote and sustain inflammation, diacerein may subsequently reduce circulating levels of inflammatory mediators.

Diacerein and apoptosis.

Diacerein was reported to induce or inhibitor apoptosis in different cell types [64–66]. Interestingly, there are reports that diacerein does not affect cell apoptosis [67, 68]. In our experimental setting, we found that diacerein inhibits apoptosis in keratinocytes while it has no role in endothelial cell apoptosis (Fig 5). The molecular mechanisms for diacerein’s role in apoptosis of keratinocytes and endothelial cells need further investigation.

thumbnail
Download:
Fig 5. Diacerein inhibits apoptosis in keratinocytes but not in endothelial cells.

Primary KCs or ECs were treated with IL-1β (10ng/ml), diacerein (20 μM for KCs and 50 μm for ECs) or both for 24 hours. Apoptosis was measured by Pan-Caspase In Situ Assay Kit as described in Methods. Values are expressed as the mean ± SEM (n = 8). * P < 0.05 vs. control.

https://doi.org/10.1371/journal.pone.0173981.g005

Conclusions

Our results show that diacerein significantly opposes pro-atherogenic and pro-inflammatory effects of IL-1 on multiple genes in ECs and KCs. Psoriasis has increased IL-1 activity and increased risk of atherosclerosis and cardiovascular morbidity [3, 12]. Because our EC data and previous studies have demonstrated that IL-1α and IL-1β contribute to vascular inflammation/atherogenesis [13–15], the elevated activity of IL-1 in psoriasis may contribute to this increased risk of atherosclerosis. Additionally, our KC data show that diacerein may diminish skin inflammation but also may have potential implications for atherogenesis in psoriasis: diacerein inhibits IL-1-induced regulation of several inflammatory genes in KCs reported to have pro-atherogenic properties, downstream in the vasculature. We therefore propose a novel idea that diacerein may act to diminish IL-1-induced atherogenesis both indirectly via skin and directly on ECs in the vasculature. Interestingly, there is a recombinant, non-glycosylated antagonist of the human IL-1 receptor, anakinra. Although limited clinical data exists, one pilot study showed anakinra to have modest benefit in patients with psoriasis and psoriatic arthritis [69]. In conclusion, this study provides evidence that diacerein reverses the pro-atherogenic and pro-inflammatory gene regulation caused by IL-1 in ECs and KCs, potentially preventing progression of skin inflammation and inflammation-induced atherosclerosis. Diacerein may have an advantage over biologic therapies. Biologics like TNF-alpha inhibitors are proteins with a partial animal component and have the potential to induce an antibody response in patients, leading to the drug’s ineffectiveness. Diacerein, being a small-molecule drug administered orally, has a substantially reduced tendency to induce such antibodies. Future investigation into clinical use of diacerein to benefit both the skin and vasculature by diminishing atherogenesis and inflammation, is warranted.

Methods

Materials

IL-1α, IL-1β, 10% fetal-bovine-serum growth medium for KCs, penicillin/streptomycin, sodium pyruvate, non-essential amino acids (Invitrogen, Carlsbad, CA), culture medium and cell-lysis kit for ECs (Cell Applications, San Diego, CA), RNA isolation kit and protocol, First strand kit, RT2 SYBR Green ROX qPCR master mix, Human Inflammatory Response and Autoimmunity 384HT PCR-arrays, Human Atherosclerosis 96-well PCR-arrays (Qiagen, Valencia, CA) (Table 5) and diacerein (lyophilized powder dissolved in 50% DMSO and filtered water) (Sigma-Aldrich, St. Louis, MO) were used.

thumbnail
Download:
Table 5. List of 84 atherosclerosis-related genes and 370 inflammation-related genes tested through PCR array

https://doi.org/10.1371/journal.pone.0173981.t005

Cell culture and treatment

Human primary keratinocytes (Invitrogen, Carlsbad, CA, catalog# C-005-25P-A) and human primary coronary artery endothelial cells (Cell Applications Inc., San Diego, CA, catalog# 300K-05a) were cultured under standard conditions (humidified atmosphere, 5% CO2 at 37°C) in growth medium supplemented with sodium pyruvate, non-essential amino acids and penicillin/streptomycin; media were replaced every 48 hours. KCs were differentiated from basal to mature keratinocytes by placing into a higher-calcium medium for 48–72 hours. At 80% confluence, cells were treated for 24 hours either with IL-1α or IL-1β only (25ng/ml IL-1α or 10ng/ml IL-1β in KCs and 10ng/ml IL-1α or 10ng/ml IL-1β in ECs), IL-1α or IL-1β combined with diacerein (diacerein concentrations: 10μM in KCs and 15μM in ECs with IL-1α; 20μM in KCs and 50μM in ECs with IL-1β–these concentrations were established using a dose-response curve prior to the experiment), or vehicle (DMSO). After 24 hours, cell culture supernatant was collected and stored at -80°C, and cells were washed with ice-cold phosphate-buffered-saline and frozen at -80°C until RNA isolation.

RNA isolation and real-time polymerase chain reaction (PCR) analysis

Total RNA from KCs and ECs were isolated using Trizol reagent per manufacturer’s instructions. Reverse-transcription using First strand kit formed cDNA. Genes reported in the literature to be regulated by IL-1 and relevant to the pathogenesis of skin or vascular inflammation/atherosclerosis, were tested by real-time PCR using custom primers (Integrated DNA Technologies, Coralville, IA).

PCR-array

Using KC and EC cDNA, PCR-arrays were used per manufacturer’s instructions. 10μL of master mix with cDNA was loaded into each well, and PCR reaction was run (ViiA-7 PCR machine, Applied Biosystems, Waltham, MA). Cycling conditions used: 95°C (10 minutes), then 40 repeating cycles of 95°C (15 seconds) followed by 60°C (60 seconds). Experiments were repeated in triplicate using four biologic replicates. Raw data were analyzed by a web-based Array Analysis Software (Qiagen). Data was normalized using house-keeping genes (ACTB, B2M, GAPDH, HPRT1, RPLP0). We then identified genes that were at least 2-fold up- or down-regulated after addition of IL-1 and/or diacerein in comparison to control, with p-value<0.05.

Enzyme linked immunosorbent assay (ELISA)

ELISA-kits were purchased based on PCR-array result. Kit were used according to manufacturer’s instructions. ELISA micro-plates were analyzed using Synergy HT Multi-Mode Microplate Reader (BioTek, Winooski, VT) per the kit protocol, usually 450nm.

For IL-1α-treated cells, CSF2 (Sigma-Aldrich), SELE (Sigma-Aldrich), IL-9 (Sigma-Aldrich), CXCR3 (MyBioSource.com, San Diego, CA), TGF-B2 (Abcam, Cambridge, MA) and ACE (Abcam) human ELISA-kits were used.

For IL-1β-treated cells, E-Selectin (Sigma-Aldrich), TNF-alpha (Invitrogen) CCL5 (Biomatic USCN Life Sciences Inc., Wuhan, Hubei, China) VCAM-1 (Sigma-Aldrich) and CSF2 (Sigma-Aldrich) human ELISA-kits were used.

Pan-caspase assay

Pan-caspase assay was performed using CaspaTag™ Pan-Caspase In Situ Assay Kit (EMD Millipore, Billerica, MA) according to the manufacturer’s instructions. Briefly, cells were seeded in 12-well plates and harvested 24 hours after treatment. Cells were trypsinized and centrifuged after trypsin inactivation. Next 90 μl of 1% charcoal-dextran-treated FBS was added into each tube with 3 μl of 30X FLICA to resuspend cells. Then cells were incubated for 1 hour at 37°C under 5% CO2. After a series of washes, cells were resuspended in PBS and counted. Absorbance using an excitation wavelength of 485 nm and an emission wavelength of 520 nm was read in a Tecan Infinite 200 Pro multimode reader (Tecan US Inc., Morrisville, NC).

Statistical analysis

PCR-array raw data were analyzed as listed above. Other data reported in this study (ELISA, real-time PCR) are the mean of 4 biologic replicates +/- standard error of the mean and were analyzed by ANOVA with p-value <0.05 taken as significant.

Supporting information

S1 File. This includes original PCR array data from endothelial cells.

https://doi.org/10.1371/journal.pone.0173981.s001

(XLS)

S2 File. This includes original PCR array data from keratinocytes.

https://doi.org/10.1371/journal.pone.0173981.s002

(XLS)

Acknowledgments

We thank Cecilia Chau for the technical support.

Presented as abstracts at the 2013 International Investigative Dermatology Meeting, Edinburg, Scotland; and 2014 Annual Meeting of the Society for Investigative Dermatology, Albuquerque, New Mexico, USA:

  1. Zhang H, Bao L, Chan LS. An IL-1β inhibitor diacerein blocks the pro-inflammatory and atherogenic effects of IL-1β on epidermal and endothelial cells. Journal of Investigative Dermatology 2013 May. 133 (S1): S17-S55, No. 151.
  2. Mohan G, Bao L, Chan LS. Diacerein inhibits pro-inflammatory and atherogenic effects of IL-1α in primary keratinocytes and endothelial cells. Journal of Investigative Dermatology. 2014 Aug. 134 (8): S4, No. LB812.

Author Contributions

  1. Conceptualization: LSC LB.
  2. Formal analysis: GCM HZ LB.
  3. Funding acquisition: LSC LB.
  4. Investigation: GCM HZ BM.
  5. Methodology: LSC LB.
  6. Project administration: LSC.
  7. Resources: LSC LB.
  8. Supervision: LSC.
  9. Validation: LB.
  10. Visualization: GCM HZ.
  11. Writing – original draft: GCM HZ.
  12. Writing – review & editing: GCM HZ LB LSC.

References

  1. 1. Parisi R, Symmons DP, Griffiths CE, Ashcroft DM. Global epidemiology of psoriasis: a systematic review of incidence and prevalence. The Journal of investigative dermatology. 2013;133(2):377–85. Epub 2012/09/28. pmid:23014338
  2. 2. Ahlehoff O, Gislason G, Lamberts M, Folke F, Lindhardsen J, Larsen CT, et al. Risk of thromboembolism and fatal stroke in patients with psoriasis and non-valvular atrial fibrillation: a Danish nationwide cohort study. Journal of internal medicine. 2014. Epub 2014/05/28.
  3. 3. Gelfand JM, Neimann AL, Shin DB, Wang X, Margolis DJ, Troxel AB. Risk of myocardial infarction in patients with psoriasis. JAMA the journal of the American Medical Association. 2006;296(14):1735–41. Epub 2006/10/13. pmid:17032986
  4. 4. Armstrong EJ, Harskamp CT, Armstrong AW. Psoriasis and major adverse cardiovascular events: a systematic review and meta-analysis of observational studies. Journal of the American Heart Association. 2013;2(2):e000062. Epub 2013/04/06. PubMed Central PMCID: PMCPmc3647278. pmid:23557749
  5. 5. Dowlatshahi EA, Kavousi M, Nijsten T, Ikram MA, Hofman A, Franco OH, et al. Psoriasis is not associated with atherosclerosis and incident cardiovascular events: the Rotterdam Study. The Journal of investigative dermatology. 2013;133(10):2347–54. Epub 2013/03/16. pmid:23492918
  6. 6. Johnston A, Fritz Y, Dawes SM, et al. Keratinocyte overexpression of IL-17C promotes psoriasiform skin inflammation. Journal of immunology (Baltimore, Md 1950). 2013;190(5):2252–62. Epub 2013/01/30. PubMed Central PMCID: PMCPmc3577967.
  7. 7. Guilloteau K, Paris I, Pedretti N, Boniface K, Juchaux F, Huguier V, et al. Skin Inflammation Induced by the Synergistic Action of IL-17A, IL-22, Oncostatin M, IL-1{alpha}, and TNF-{alpha} Recapitulates Some Features of Psoriasis. Journal of immunology (Baltimore, Md 1950). 2010. Epub 2010/03/26.
  8. 8. Buerger C, Richter B, Woth K, Salgo R, Malisiewicz B, Diehl S, et al. Interleukin-1beta interferes with epidermal homeostasis through induction of insulin resistance: implications for psoriasis pathogenesis. The Journal of investigative dermatology. 2012;132(9):2206–14. Epub 2012/04/20. pmid:22513786
  9. 9. Balato A, Schiattarella M, Lembo S, Mattii M, Prevete N, Balato N, et al. Interleukin-1 family members are enhanced in psoriasis and suppressed by vitamin D and retinoic acid. Archives of dermatological research. 2013;305(3):255–62. Epub 2013/02/26. pmid:23435685
  10. 10. Nestle FO, Kaplan DH, Barker J. Psoriasis. New England Journal of Medicine. 2009;361(5):496–509. pmid:19641206
  11. 11. Libby P. Inflammation in atherosclerosis. Arteriosclerosis, thrombosis, and vascular biology. 2012;32(9):2045–51. Epub 2012/08/17. PubMed Central PMCID: PMCPmc3422754. pmid:22895665
  12. 12. Wakkee M, Thio HB, Prens EP, Sijbrands EJ, Neumann HA. Unfavorable cardiovascular risk profiles in untreated and treated psoriasis patients. Atherosclerosis. 2007;190(1):1–9. Epub 2006/09/01. pmid:16942772
  13. 13. Kaplan MJ. Cardiometabolic risk in psoriasis: differential effects of biologic agents. Vascular health and risk management. 2008;4(6):1229–35. Epub 2008/01/01. PubMed Central PMCID: PMCPmc2663453. pmid:19337536
  14. 14. Freigang S, Ampenberger F, Weiss A, Kanneganti TD, Iwakura Y, Hersberger M, et al. Fatty acid-induced mitochondrial uncoupling elicits inflammasome-independent IL-1alpha and sterile vascular inflammation in atherosclerosis. Nature immunology. 2013;14(10):1045–53. Epub 2013/09/03. pmid:23995233
  15. 15. Beaulieu LM, Lin E, Mick E, et al. Interleukin 1 receptor 1 and interleukin 1beta regulate megakaryocyte maturation, platelet activation, and transcript profile during inflammation in mice and humans. Arteriosclerosis, thrombosis, and vascular biology. 2014;34(3):552–64. Epub 2014/01/25. pmid:24458711
  16. 16. Xie W, Liu J, Wang W, Wang M, Li Y, Sun J, et al. Five-year change in systolic blood pressure is independently associated with carotid atherosclerosis progression: a population-based cohort study. Hypertension research official journal of the Japanese Society of Hypertension. 2014. Epub 2014/05/09.
  17. 17. Li JJ, Chen JL. Inflammation may be a bridge connecting hypertension and atherosclerosis. Medical hypotheses. 2005;64(5):925–9. Epub 2005/03/23. pmid:15780486
  18. 18. Chang CH, Huang Y, Anderson R. Activation of vascular endothelial cells by IL-1alpha released by epithelial cells infected with respiratory syncytial virus. Cellular immunology. 2003;221(1):37–41. Epub 2003/05/14. pmid:12742380
  19. 19. Fonsatti E, Altomonte M, Coral S, Cattarossi I, Nicotra MR, Gasparollo A, et al. Tumour-derived interleukin 1alpha (IL-1alpha) up-regulates the release of soluble intercellular adhesion molecule-1 (sICAM-1) by endothelial cells. British journal of cancer. 1997;76(10):1255–61. Epub 1997/01/01. PubMed Central PMCID: PMCPmc2228138. pmid:9374368
  20. 20. Kitagawa K, Matsumoto M, Sasaki T, Hashimoto H, Kuwabara K, Ohtsuki T, et al. Involvement of ICAM-1 in the progression of atherosclerosis in APOE-knockout mice. Atherosclerosis. 2002;160(2):305–10. Epub 2002/02/19. pmid:11849652
  21. 21. Nageh MF, Sandberg ET, Marotti KR, Lin AH, Melchior EP, Bullard DC, et al. Deficiency of inflammatory cell adhesion molecules protects against atherosclerosis in mice. Arteriosclerosis, thrombosis, and vascular biology. 1997;17(8):1517–20. Epub 1997/08/01. pmid:9301629
  22. 22. Hanemaaijer R, Koolwijk P, le Clercq L, de Vree WJ, van Hinsbergh VW. Regulation of matrix metalloproteinase expression in human vein and microvascular endothelial cells. Effects of tumour necrosis factor alpha, interleukin 1 and phorbol ester. The Biochemical journal. 1993;296 Pt 3):803–9. Epub 1993/12/15. PubMed Central PMCID: PMCPmc1137766.
  23. 23. Nikkari ST, O'Brien KD, Ferguson M, Hatsukami T, Welgus HG, Alpers CE, et al. Interstitial collagenase (MMP-1) expression in human carotid atherosclerosis. Circulation. 1995;92(6):1393–8. Epub 1995/09/15. pmid:7664418
  24. 24. Lehrke M, Greif M, Broedl UC, Lebherz C, Laubender RP, Becker A, et al. MMP-1 serum levels predict coronary atherosclerosis in humans. Cardiovascular diabetology. 2009;8:50. Epub 2009/09/16. PubMed Central PMCID: PMCPmc2754422. pmid:19751510
  25. 25. Dollery CM, Libby P. Atherosclerosis and proteinase activation. Cardiovascular research. 2006;69(3):625–35. Epub 2005/12/27. pmid:16376322
  26. 26. Son SH, Goo YH, Chang BH, Paul A. Perilipin 2 (PLIN2)-deficiency does not increase cholesterol-induced toxicity in macrophages. PloS one. 2012;7(3):e33063. Epub 2012/03/20. PubMed Central PMCID: PMCPmc3299742. pmid:22427949
  27. 27. Etter H, Althaus R, Eugster HP, Santamaria-Babi LF, Weber L, Moser R. IL-4 and IL-13 downregulate rolling adhesion of leukocytes to IL-1 or TNF-alpha-activated endothelial cells by limiting the interval of E-selectin expression. Cytokine. 1998;10(6):395–403. Epub 1998/06/25. pmid:9632524
  28. 28. Dong ZM, Chapman SM, Brown AA, Frenette PS, Hynes RO, Wagner DD. The combined role of P- and E-selectins in atherosclerosis. The Journal of clinical investigation. 1998;102(1):145–52. Epub 1998/07/03. PubMed Central PMCID: PMCPmc509076. pmid:9649568
  29. 29. Galkina E, Ley K. Vascular adhesion molecules in atherosclerosis. Arteriosclerosis, thrombosis, and vascular biology. 2007;27(11):2292–301. Epub 2007/08/04. pmid:17673705
  30. 30. Tregouet DA, Barbaux S, Poirier O, Blankenberg S, Bickel C, Escolano S, et al. SELPLG Gene Polymorphisms in Relation to Plasma SELPLG Levels and Coronary Artery Disease. Annals of Human Genetics. 2003;67(6):504–11.
  31. 31. Wolfrum S, Teupser D, Tan M, Chen KY, Breslow JL. The protective effect of A20 on atherosclerosis in apolipoprotein E-deficient mice is associated with reduced expression of NF-kappaB target genes. Proceedings of the National Academy of Sciences of the United States of America. 2007;104(47):18601–6. Epub 2007/11/17. PubMed Central PMCID: PMCPmc2141823. pmid:18006655
  32. 32. Rolfe BE, Stamatiou S, World CJ, Brown L, Thomas AC, Bingley JA, et al. Leukaemia inhibitory factor retards the progression of atherosclerosis. Cardiovascular research. 2003;58(1):222–30. Epub 2003/04/02. pmid:12667965
  33. 33. Moran CS, Campbell JH, Simmons DL, Campbell GR. Human leukemia inhibitory factor inhibits development of experimental atherosclerosis. Arteriosclerosis and thrombosis a journal of vascular biology / American Heart Association. 1994;14(8):1356–63. Epub 1994/08/01.
  34. 34. Barlic J, Murphy PM. Chemokine regulation of atherosclerosis. Journal of leukocyte biology. 2007;82(2):226–36. Epub 2007/03/03. pmid:17329566
  35. 35. Wong D, Dorovini-Zis K. Expression of vascular cell adhesion molecule-1 (VCAM-1) by human brain microvessel endothelial cells in primary culture. Microvascular research. 1995;49(3):325–39. Epub 1995/05/01. pmid:7543972
  36. 36. Cuff CA, Kothapalli D, Azonobi I, Chun S, Zhang Y, Belkin R, et al. The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation. The Journal of clinical investigation. 2001;108(7):1031–40. Epub 2001/10/03. PubMed Central PMCID: PMCPmc200948. pmid:11581304
  37. 37. Golledge J, Clancy P, Maguire J, Lincz L, Koblar S. The role of tenascin C in cardiovascular disease. Cardiovascular research. 2011;92(1):19–28. Epub 2011/06/30. pmid:21712412
  38. 38. Ranta V, Orpana A, Carpen O, Turpeinen U, Ylikorkala O, Viinikka L. Human vascular endothelial cells produce tumor necrosis factor-alpha in response to proinflammatory cytokine stimulation. Critical care medicine. 1999;27(10):2184–7. Epub 1999/11/05. pmid:10548204
  39. 39. Ait-Oufella H, Taleb S, Mallat Z, Tedgui A. Recent advances on the role of cytokines in atherosclerosis. Arteriosclerosis, thrombosis, and vascular biology. 2011;31(5):969–79. Epub 2011/04/22. pmid:21508343
  40. 40. Levula M, Oksala N, Airla N, et al. Genes involved in systemic and arterial bed dependent atherosclerosis—Tampere Vascular study. PloS one. 2012;7(4):e33787. Epub 2012/04/18. PubMed Central PMCID: PMCPmc3324479. pmid:22509262
  41. 41. Grainger DJ. TGF-beta and atherosclerosis in man. Cardiovascular research. 2007;74(2):213–22. Epub 2007/03/27. pmid:17382916
  42. 42. Cataisson C, Pearson AJ, Tsien MZ, Mascia F, Gao J-L, Pastore S, et al. CXCR2 ligands and G-CSF mediate PKCα-induced intraepidermal inflammation. The Journal of clinical investigation. 2006;116(10):2757–66. pmid:16964312
  43. 43. Butcher M, Galkina E. Murine aortic vascular cells produce IL-17C and support inflammation in atherosclerosis. (CCR6P.271). The Journal of Immunology. 2014;192(1 Supplement):182.3.
  44. 44. Iwakura Y, Ishigame H. The IL-23/IL-17 axis in inflammation. The Journal of clinical investigation. 2006;116(5):1218–22. Epub 2006/05/04. PubMed Central PMCID: PMCPmc1451213. pmid:16670765
  45. 45. Gao Q, Jiang Y, Ma T, et al. A critical function of Th17 proinflammatory cells in the development of atherosclerotic plaque in mice. Journal of immunology (Baltimore, Md 1950). 2010;185(10):5820–7. Epub 2010/10/19.
  46. 46. David A, Saitta S, De Caridi G, Benedetto F, Massara M, Risitano DC, et al. Interleukin-23 serum levels in patients affected by peripheral arterial disease. Clinical biochemistry. 2012;45(3):275–8. Epub 2012/01/03. pmid:22209993
  47. 47. Muhr P, Renne J, Schaefer V, Werfel T, Wittmann M. Primary human keratinocytes efficiently induce IL-1-dependent IL-17 in CCR6+ T cells. Experimental dermatology. 2010;19(12):1105–7. Epub 2010/09/04. pmid:20812962
  48. 48. Peral de Castro C, Jones SA, Ni Cheallaigh C, Hearnden CA, Williams L, Winter J, et al. Autophagy regulates IL-23 secretion and innate T cell responses through effects on IL-1 secretion. Journal of immunology (Baltimore, Md 1950). 2012;189(8):4144–53. Epub 2012/09/14.
  49. 49. Chen F, Yu Y, Haigh S, Johnson J, Lucas R, Stepp DW, et al. Regulation of NADPH Oxidase 5 by Protein Kinase C Isoforms. PloS one. 2014;9(2):e88405. pmid:24505490
  50. 50. Guzik TJ, Chen W, Gongora MC, Guzik B, Lob HE, Mangalat D, et al. Calcium-dependent NOX5 nicotinamide adenine dinucleotide phosphate oxidase contributes to vascular oxidative stress in human coronary artery disease. Journal of the American College of Cardiology. 2008;52(22):1803–9. Epub 2008/11/22. PubMed Central PMCID: PMCPmc2593790. pmid:19022160
  51. 51. Bedard K, Jaquet V, Krause KH. NOX5: from basic biology to signaling and disease. Free radical biology & medicine. 2012;52(4):725–34. Epub 2011/12/21.
  52. 52. Zhong XF, Huang GD, Luo T, Deng ZY, Hu JN. Protective effect of rhein against oxidative stress-related endothelial cell injury. Molecular medicine reports. 2012;5(5):1261–6. Epub 2012/02/22. pmid:22344690
  53. 53. Liu L, Shi GP. CD31: beyond a marker for endothelial cells. Cardiovascular research. 2012;94(1):3–5. Epub 2012/03/02. pmid:22379038
  54. 54. Swindell WR, Johnston A, Xing X, Voorhees JJ, Elder JT, Gudjonsson JE. Modulation of epidermal transcription circuits in psoriasis: new links between inflammation and hyperproliferation. PloS one. 2013;8(11):e79253. Epub 2013/11/22. PubMed Central PMCID: PMCPmc3829857. pmid:24260178
  55. 55. Romacho T, Sanchez-Ferrer CF, Peiro C. Visfatin/Nampt: an adipokine with cardiovascular impact. Mediators Inflamm. 2013;2013:946427. Epub 2013/07/12. PubMed Central PMCID: PMCPmc3697395. pmid:23843684
  56. 56. Kim WJ, Bae EM, Kang YJ, Bae HU, Hong SH, Lee JY, et al. Glucocorticoid-induced tumour necrosis factor receptor family related protein (GITR) mediates inflammatory activation of macrophages that can destabilize atherosclerotic plaques. Immunology. 2006;119(3):421–9. Epub 2006/10/28. PubMed Central PMCID: PMCPmc1819571. pmid:17067317
  57. 57. Hu Z, Zhang J, Guan A, Gong H, Yang M, Zhang G, et al. Granulocyte colony-stimulating factor promotes atherosclerosis in high-fat diet rabbits. International journal of molecular sciences. 2013;14(3):4805–16. Epub 2013/03/02. PubMed Central PMCID: PMCPmc3634472. pmid:23449031
  58. 58. Yao Y, Bennett BJ, Wang X, Rosenfeld ME, Giachelli C, Lusis AJ, et al. Inhibition of bone morphogenetic proteins protects against atherosclerosis and vascular calcification. Circulation research. 2010;107(4):485–94. Epub 2010/06/26. PubMed Central PMCID: PMCPmc2994650. pmid:20576934
  59. 59. Zhou B, Margariti A, Zeng L, Xu Q. Role of histone deacetylases in vascular cell homeostasis and arteriosclerosis. Cardiovascular research. 2011;90(3):413–20. Epub 2011/01/15. pmid:21233251
  60. 60. Nold-Petry CA, Nold MF, Zepp JA, Kim S- H, Voelkel NF, Dinarello CA. IL-32–dependent effects of IL-1β on endothelial cell functions. Proceedings of the National Academy of Sciences. 2009;106(10):3883–8.
  61. 61. Pirillo A, Catapano AL. Soluble lectin-like oxidized low density lipoprotein receptor-1 as a biochemical marker for atherosclerosis-related diseases. Disease markers. 2013;35(5):413–8. Epub 2013/11/08. PubMed Central PMCID: PMCPmc3809739. pmid:24198442
  62. 62. Kim YJ, Koo TY, Yang WS, Han NJ, Jeong JU, Lee SK, et al. Activation of spleen tyrosine kinase is required for TNF-alpha-induced endothelin-1 upregulation in human aortic endothelial cells. FEBS letters. 2012;586(6):818–26. Epub 2012/02/11. pmid:22321643
  63. 63. Hilgendorf I, Eisele S, Remer I, et al. The oral spleen tyrosine kinase inhibitor fostamatinib attenuates inflammation and atherogenesis in low-density lipoprotein receptor-deficient mice. Arteriosclerosis, thrombosis, and vascular biology. 2011;31(9):1991–9. Epub 2011/06/28. pmid:21700926
  64. 64. Bharti R, Dey G, Ojha PK, Rajput S, Jaganathan SK, Sen R, et al. Diacerein-mediated inhibition of IL-6/IL-6R signaling induces apoptotic effects on breast cancer. Oncogene. 2016;35(30):3965–75. pmid:26616855
  65. 65. Li ZF, Meng DD, Li GH, Xu JZ, Tian K, Li Y. Celecoxib Combined with Diacerein Effectively Alleviates Osteoarthritis in Rats via Regulating JNK and p38MAPK Signaling Pathways. Inflammation. 2015;38(4):1563–72. pmid:25687638
  66. 66. Li H, Liang C, Chen Q, Yang Z. Rhein: a potential biological therapeutic drug for intervertebral disc degeneration. Med Hypotheses. 2011;77(6):1105–7. pmid:21962453
  67. 67. Legendre F, Heuze A, Boukerrouche K, Leclercq S, Boumediene K, Galera P, et al. Rhein, the metabolite of diacerhein, reduces the proliferation of osteoarthritic chondrocytes and synoviocytes without inducing apoptosis. Scand J Rheumatol. 2009;38(2):104–11. pmid:19274517
  68. 68. Lohberger B, Leithner A, Stuendl N, Kaltenegger H, Kullich W, Steinecker-Frohnwieser B. Diacerein retards cell growth of chondrosarcoma cells at the G2/M cell cycle checkpoint via cyclin B1/CDK1 and CDK2 downregulation. Bmc Cancer. 2015;15.
  69. 69. Gibbs A, Markham T, Walsh C, Bresnihan B, Veale D, FitzGerald O. Anakinra (Kineret) in psoriasis and psoriatic arthritis: a single-center, open-label, pilot study. Arthritis Res Ther. 2005;7:S27-S.