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Inhibitory effects of harpagoside on TNF-α-induced pro-inflammatory adipokine expression through PPAR-γ activation in 3T3-L1 adipocytes
Cytokine xxx (2015) xxx–xxx
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Inhibitory effects of harpagoside on TNF-a-induced pro-inflammatory adipokine expression through PPAR-c activation in 3T3-L1 adipocytes Tae Kon Kim a, Kyoung Sik Park b,⇑ a b
Department of Medicinal Chemistry, College of Science and Engineering, Jungwon University, Republic of Korea Department of Biomedical Science, College of Science and Engineering, Cheongju University, Chungbuk, Republic of Korea
a r t i c l e
i n f o
Article history: Received 10 April 2015 Received in revised form 15 May 2015 Accepted 15 May 2015 Available online xxxx Keywords: Iridoid glycoside Herbal medicine Adipocyte Pro-inflammatory adipokine Atherosclerosis
a b s t r a c t Obesity is closely associated with increased production of pro-inflammatory adipokines, including interleukin (IL)-6, plasminogen activator inhibitor (PAI)-1, and adipose-tissue-derived monocyte chemoattractant protein (MCP)-1, which contribute to chronic and low-grade inflammation in adipose tissue. Harpagoside, a major iridoid glycoside present in devil’s claw, has been reported to show anti-inflammatory activities by suppression of lipopolysaccharide (LPS)-induced production of inflammatory cytokines in murine macrophages. The present study is aimed to investigate the effects of harpagoside on both tumor necrosis factor (TNF)-a-induced inflammatory adipokine expression and its underlying signaling pathways in differentiated 3T3-L1 cells. Harpagoside significantly inhibited TNF-a-induced mRNA synthesis and protein production of the atherogenic adipokines including IL-6, PAI-1, and MCP-1. Further investigation of the molecular mechanism revealed that pretreatment with harpagoside activated peroxisome proliferator–activated receptor (PPAR)-c. These findings suggest that the clinical application of medicinal plants which contain harpagoside may lead to a partial prevention of obesity-induced atherosclerosis by attenuating inflammatory responses. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Harpagoside is a naturally occurring iridoid glycoside found in many medicinal plants such as Scrophularia ningpoensis, Scrophularia buergeriana, and Harpagophytum procumbens [1–3]. These medicinal plants have been shown to exhibit a variety of biological activities and used as pharmaceutical products for the treatment of inflammatory ailment, rheumatoid arthritis, and osteoarthritis [4–6]. Harpagoside is believed to be a main bioactive compound related to the anti-inflammatory efficacy of these plants, and the harpagoside content is used to standardize commercial H. procumbens products, which should contain at least 1.2% of the compound according to the European Pharmacopoeia [7]. As for the molecular mechanism for anti-inflammatory activities of harpagoside, it was reported that harpagoside inhibits lipopolysaccharide (LPS)-induced production of inflammatory cytokines such as interleukin (IL)-1b, IL-6, and tumor necrosis factor (TNF)-a resulting from the both IjBa degradation and the
⇑ Corresponding author at: Department of Biomedical Science, College of Science and Engineering, Cheongju University, 298 Daesung-ro, Chungwon-gu, Cheongju-si, Chungbuk 363-764, Republic of Korea. Tel.: +82 43 229 8566; fax: +82 43 229 8559. E-mail address: [email protected] (K.S. Park).
nuclear translocation of NF-jB in RAW 264.7 cells [8]. However, the effects of harpagoside on obesity-related inflammatory responses in adipocytes have not been reported previously. Obesity, which is characterized by excessive accumulation of abdominal fat, is casually associated with the premature development of atherosclerosis, increased risk of stroke, and development of congestive heart failure [9]. Recent studies have indicated that the adipocyte secretes a variety of adipokines involved in energy metabolism, inflammation, and cardiovascular functions [10]. The cellular mechanisms linking obesity and atherosclerosis are complex and have not been fully elucidated. However, increasing evidences suggest that the changes of adipokines including IL-6, plasminogen activator inhibitor (PAI)-1, and monocyte chemoattractant protein (MCP)-1 due to excess adipose tissue may be a cause of atherosclerosis [11]. IL-6 is produced by many cell types and many tissues including adipose tissue. It is now well known that IL-6 production by adipose tissue is enhanced in obesity [12,13]. IL-6 has been recently proposed to play a central role in the link between obesity, inflammation, and coronary heart diseases [14]. PAI-1 is the primary inhibitor of plasminogen activation. Plasma levels of PAI-1 are markedly elevated in obese individuals as well as in patients with insulin resistance, type 2 diabetes, and cardiovascular diseases
http://dx.doi.org/10.1016/j.cyto.2015.05.015 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kim TK, Park KS. Inhibitory effects of harpagoside on TNF-a-induced pro-inflammatory adipokine expression through PPAR-c activation in 3T3-L1 adipocytes. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.05.015
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T.K. Kim, K.S. Park / Cytokine xxx (2015) xxx–xxx
(CVDs) [15,16]. PAI-1 is thought to be the link between obesity and increased risk for CVDs [17]. Although several tissues are known to produce PAI-1, adipose tissue appears to be the major contributor to elevated PAI-1 levels observed in cases of obesity [18,19]. Monocyte chemoattractant protein (MCP)-1 chemotactically recruits monocytes to sites of inflammation. Although this protein is traditionally thought to be expressed mainly endothelial cells and macrophages, it has been shown to be primarily expressed by adipose tissues [20,21]. Recently, MCP-1 has been reported to be a novel adipokine involved in the development of obesity-related insulin resistance and atherosclerosis [22]. The present study was designed to determine whether harpagoside attenuates TNF-a-induced secretion and mRNA production of the atherogenic adipokines including IL-6, PAI-1, and MCP-1 in differentiated 3T3-L1 adipocytes. In addition, the possible mechanisms for inhibitory effects of harpagoside on obesity-related inflammatory responses were examined.
2. Materials and methods
2.4. Evaluation of gene expression levels by quantitative real-time RTPCR For real-time RT-PCR, cDNA was prepared from 1 lg total RNA in a final reaction volume of 15 lL. Real-time RT-PCR was performed using 2 lL cDNA diluted with each of the gene-specific primer sets (Bioneer Inc., Korea). Each primer set was used at a concentration of 150 nM in a final reaction volume of 20 lL and reactions were performed on the Lightcycler 480 SYBR Green I Master (Roche Applied Science, Germany). The sequences for primers are: IL-6 (F: 50 -GCTACCAAACTGGATATAATCAGGA-30 and R: 50 -CCAGGTAGCTATGGTACTCCAGAA-30 ), PAI-1 (F: 50 -AGGATCGAG GTAAACGAGAGC-30 and R: 50 -GCGGGCTGAGATGACAAA-30 ), MCP-1 (F: 50 -GCCCCACTCACCTGCTGCTACT-30 and R: 50 -CCTGCTG CTGGTGATCCTCTTGT-30 ), aP2 (F: 50 -TGAAAGAAGTAGGAGTGGG C-30 and R: 50 -CTTCAGTCCAGGTCAACGTC-30 ), adiponectin (F: 50 -G TTCTACTGCAACATTCCGG-30 and R: 50 -TACACCTGGAGCCAGACTT G-30 ) and GAPDH (F: 50 -CGGTGCTGAGTATGTCTGTG-30 and R: 50 -G GTGGAGATGATGACCCTTT-30 ) [23,24]. All quantifications were performed in duplicate and the experiments were independently repeated three times.
2.1. Reagents Harpagoside with higher than 99.0% purity was purchased from Extrasynthese (Genay, France). Recombinant murine tumor necrosis factor (TNF)-a was from R&D Systems (Minneapolis, MN, USA). Insulin, dexamethasone, 3-isobutyl-1-methylxanthine, and GW9662 (2-chloro-5-nitrobenzanilide) were from Sigma (St. Louis, MO, USA). All tissue culture materials were from Gibco-BRL (Rockville, MD, USA). All the other chemicals used were purchased from Sigma (St. Louis, MO, USA). 2.2. 3T3-L1 cell culture and cytotoxicity test 3T3-L1 preadipocytes (American Type Culture Collection, Rockville, MD, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin–streptomycin (10,000 U/mL penicillin and 10,000 lg/mL streptomycin in 0.85% saline), and 1% (v/v) 100 lM pyruvate at 37 °C in 95% air 10% CO2. Differentiation of 2-day postconfluent preadipocytes (designated as day 0) was initiated with 0.5 mM 3-isobutyl-1-methylxanthine, 1 lM dexamethasone, and 1 lg/mL insulin in DMEM supplemented with 10% fetal bovine serum. After 48 h (day 2), the culture medium was replaced with DMEM supplemented with 10% fetal bovine serum and 1 lg/mL insulin, and the cells were then fed every other day with DMEM containing 10% fetal bovine serum. Harpagoside was reconstituted, filter sterilized, and stored at 20 °C. For each experiment, cells received harpagoside premixed with culture medium. TNF-a treatment was carried out after a 6-h pretreatment with harpagoside. Harpagoside was dissolved in dimethyl sulfoxide (DMSO) before it was added to culture medium. The final concentration of DMSO in culture medium was 0.1% (v/v). Possible cytotoxicity of harpagoside and TNF-a was assessed by the 3-(4,5-dimethylthiazo l-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) staining method. 2.3. Measurement of adipokines by ELISA The conditioned culture medium from 3T3-L1 adipocytes was collected from each sample. The concentrations of IL-6, PAI-1, and MCP-1 were assayed using a mouse IL-6 ELISA kit (Biosource Inc., Camarillo, CA, USA), a mouse PAI-1 ELISA kit (Molecular Innovations Inc., Southfield, MI, USA), and a mouse MCP-1 ELISA kit (R&D Systems, Minneapolis, MN, USA), respectively. All assays were performed according to the manufacturer’s instructions.
2.5. Luciferase reporter assay Luciferase assay was performed using the dual luciferase system (Promega, Madison, WI, USA) according to the manufacturer’s protocols. For PPAR-c luciferase assay, p4xUASg-tk-luc (a reporter plasmid), pRL-CMV (an internal control) and pM-PPAR-c (an expression plasmid for GAL4/PPAR-ligand binding domain chimera protein) were transfected into CV1 cells cultured on 24-well tissue culture plates. The transfection was performed by LipofectAMINE (Invitrogen, Carlsbad, CA, USA) using the manufacturer’s instructions. Twenty-four hours after the transfection, the transfected cells were cultured in medium containing harpagoside for an additional 24 h. 2.6. Statistics Data expressed as mean ± SD were obtained from three separate experiments. Statistical analysis of the data was performed by ANOVA and the unpaired t-test. P < 0.05 was considered to have significant difference. 3. Results 3.1. No cytotoxic effects of harpagoside and TNF-a on 3T3-L1 adipocytes We performed MTT test to evaluate the possibility of cytotoxicity of harpagoside and TNF-a on adipocytes that could misinterpret results. As shown in Fig. 1, harpagoside in concentrations of 10–100 lM with/without 10 ng/mL TNF-a did not show significant cytotoxicity. The relative viability of adipocytes was all greater than 95% when measured at 96 h after treatment. 3.2. Effects of harpagoside on the TNF-a-induced secretion of adipokines in 3T3-L1 adipocytes To examine the effects of harpagoside on TNF-a-induced secretion of adipokines, 3T3-L1 adipocytes were pretreated with various concentrations of harpagoside for 6 h and then incubated with 10 ng/mL TNF-a for 24 h. After incubation, conditioned medium was collected for ELISA assay. Treatment with harpagoside inhibited TNF-a-induced increase in the secretion of IL-6, PAI-1, and
Please cite this article in press as: Kim TK, Park KS. Inhibitory effects of harpagoside on TNF-a-induced pro-inflammatory adipokine expression through PPAR-c activation in 3T3-L1 adipocytes. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.05.015
T.K. Kim, K.S. Park / Cytokine xxx (2015) xxx–xxx
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Fig. 1. No cytotoxicity of harpagoside and TNF-a to 3T3-L1 adipocytes. 3T3-L1 cells were incubated with increasing concentrations of harpagoside (from 10 to 100 lM, white bars) or with TNF-a (10 ng/mL, black bars) for 96 h. The cell viability was then determined by the MTT assay. Cell viability was normalized with that of untreated control (DMSO only treated) and is shown as percentages of relative cell viability. Values are mean ± SD of three independent experiments.
Fig. 2. Effects of harpagoside on TNF-a-stimulated adipokine production in 3T3-L1 adipocytes. 3T3-L1 adipocytes were exposed or not to increasing concentrations of harpagoside (5, 10, 20 and 50 lM) for 6 h and then were stimulated for 24 h by the addition of 10 ng/mL TNF-a. The secreted antigens of IL-6, PAI-1, and MCP-1 in conditioned medium were measured as described in Materials and Methods. Results represent the mean ± SD from three independent experiments. ##p < 0.01 versus untreated control; ⁄ p < 0.05, ⁄⁄p < 0.01 versus TNF-a-stimulated control.
Please cite this article in press as: Kim TK, Park KS. Inhibitory effects of harpagoside on TNF-a-induced pro-inflammatory adipokine expression through PPAR-c activation in 3T3-L1 adipocytes. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.05.015
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MCP-1 in dose-dependent manner with IC50 of 14.04 ± 2.60, 18.68 ± 0.48, and 12.18 ± 6.35 lM, respectively (Fig. 2). 3.3. Effects of harpagoside on the TNF-a-induced expression of adipokine genes in 3T3-L1 adipocytes To investigate whether harpagoside inhibited TNF-a-induced secretion of adipokines by altering the gene expression, real-time quantitative RT-PCR was carried out with total RNA extracted from 3T3-L1 adipocytes of each group. The gene expression of adipokines was measured after 3T3-L1 adipocytes were exposed to harpagoside for 6 h prior to TNF-a (10 ng/mL) treatment for 24 h. The enhanced production of IL-6, PAI-1, and MCP-1 mRNA by TNF-a was suppressed by harpagoside pretreatment (Fig. 3). At 20 lM of harpagoside, the mRNA production of IL-6, PAI-1, and MCP-1 was suppressed by 75.5%, 59.4%, and 69.5%, respectively. 3.4. Effects of harpagoside on the activity of PPAR-c To investigate the underlying mechanism of anti-inflammatory activities of harpagoside, we evaluated the effects of harpagoside on PPAR-c activity by using the luciferase ligand assay system. In assays with GAL4 and PPAR-c-chimera protein, harpagoside significantly activated GAL4/PPAR-c chimera transactivations in dose-dependent manner as shown in Fig. 4A. The level of PPAR-c activity with the treatment of 20 lM harpagoside was
approximately 2.0-fold higher than that in the vehicle control. Furthermore, to determine whether the effects of harpagoside are caused by PPAR-c activation, GW9662, a PPAR-c antagonist, was added together with harpagoside to the culture medium of 3T3-L1 adipocytes. GW9662 significantly increased both the mRNA production and protein synthesis of IL-6 and MCP-1 which had been reduced by the treatment of 20 lM harpagoside (Fig. 4B and C), indicating that PPAR-c plays an important role in the inhibition of the gene expression of the atherogenic adipokines by harpagoside. Moreover, to confirm the signaling pathway of harpagoside through PPAR-c activation, we examined the effects of harpagoside on the mRNA synthesis of PPAR-c target genes in TNF-a treated adipocytes. As shown in Fig. 5, TNF-a suppressed mRNA production of PPAR-c target genes including adipocyte fatty acid-binding protein (aP2) and adiponectin. Pretreatment of harpagoside attenuated TNF-a-mediated suppression of mRNA expressions of aP2 and adiponectin. These data indicate that harpagoside induces the expression of PPAR-c target genes through the PPAR-c-dependent pathway. 4. Discussions The anti-inflammatory activities of harpagoside have been mainly evaluated in mouse and rat carrageenan-induced oedema models [25,26]. Although the anti-inflammatory action of
Fig. 3. Effects of harpagoside on TNF-a-induced adipokine mRNA synthesis in 3T3-L1 adipocytes. After exposure to various concentrations of harpagoside (5, 10, 20 and 50 lM) for 6 h, 3T3-L1 adipocytes were stimulated with 10 ng/mL TNF-a for 24 h. Then cells were harvested, and total RNA was extracted for measuring the mRNA levels of IL-6, PAI-1, and MCP-1 by quantitative RT-PCR. Values are normalized to b-actin RNA expression levels and expressed relative to untreated control cells. Results represent the mean ± SD from three independent experiments. ##p < 0.01 versus untreated control; ⁄p < 0.05, ⁄⁄p < 0.01 versus TNF-a-stimulated control.
Please cite this article in press as: Kim TK, Park KS. Inhibitory effects of harpagoside on TNF-a-induced pro-inflammatory adipokine expression through PPAR-c activation in 3T3-L1 adipocytes. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.05.015
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Fig. 4. Effects of harpagoside on activation of PPAR-c. (A) Results of luciferase assays showing dose-dependencies of harpagoside on PPAR-c activities. p4xUASg-tk-luc and pRL-CMV were infected into CV1 cells together with pM-PPAR-c. Twenty-four hours after the infection, the cells were treated with harpagoside at various concentrations for 24 h. The activity of vehicle control was set at 100% and relative luciferase activity was presented as fold induction relative to that of the vehicle control. The values are mean ± SD of three independent experiments. ⁄⁄p < 0.01 compared with vehicle control. (B and C) Effects of PPAR-c inhibitor GW9662 on harpagoside-induced suppression of TNF-a-stimulated adipokine mRNA expression and protein synthesis in 3T3-L1 adipocytes. After exposure to 20 lM harpagoside in the absence or presence of GW9662 (10 lM) for 6 h and then were stimulated for 24 h by the addition of 10 ng/mL TNF-a. Then cells were harvested, and total RNA was extracted for measuring the mRNA levels of IL-6, PAI-1, and MCP-1 by quantitative RT-PCR. Values are normalized to b-actin RNA expression levels and expressed relative to untreated control cells (B). Moreover, the secreted antigens of IL-6, PAI-1, and MCP-1 in conditioned medium were measured as described in Materials and Methods (C). Results represent the mean ± SD from three independent experiments (a, b, and c: p < 0.05 versus each other).
harpagoside has been demonstrated in RAW 264.7 cells via NF-jB signaling pathway [8], the effects of harpagoside on obesity-related inflammatory responses in adipocytes have not been reported previously. The present study showed for the first time that harpagoside, a major component of H. procumbens, effectively suppressed the TNF-a-mediated increase in IL-6, PAI-1, and MCP-1 in 3T3-L1 adipocytes (Fig. 2). It was previously reported that the increased levels of TNF-a associated with obesity may significantly contribute to elevated plasma and adipose tissue expression levels of adipokines [27]. The TNF-a-induced changes in the mRNA expression of each adipokine were also inhibited by harpagoside (Fig. 3). These results indicate that harpagoside affects the TNF-a-mediated changes in the secretion of adipokines at the transcription level. In attempts to elucidate the possible mechanisms for inhibitory effects of harpagoside on obesity-related inflammatory responses,
it was found that harpagoside may act as an agonist for PPAR-c in luciferase ligand assays (Fig. 4A). This is confirmed by the fact that effects of harpagoside on regulation of TNF-a-induced adipokine protein production as well as mRNA expression disappeared by the addition of GW9662, a PPAR-c antagonist (Fig. 4B and C). In addition, harpagoside attenuated TNF-a-mediated suppression of mRNA expressions of PPAR-c target genes including aP2 and adiponectin, implying that harpagoside exerts the anti-inflammatory activities through the PPAR-c-dependent pathway (Fig. 5). Adiponectin expression is exclusive to adipose tissues and the mRNA expression level of adiponectin is low in obese mice and humans. Furthermore, a functional PPAR-responsive element (PPRE) has been identified in human adiponectin promoters [28]. PPAR-c promotes the transcription of adiponectin, which modulates glucose metabolism and exerts anti-inflammatory actions
Please cite this article in press as: Kim TK, Park KS. Inhibitory effects of harpagoside on TNF-a-induced pro-inflammatory adipokine expression through PPAR-c activation in 3T3-L1 adipocytes. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.05.015
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Fig. 5. Effects of harpagoside on TNF-a-induced mRNA synthesis of PPAR-c target genes in 3T3-L1 adipocytes. Real-time PCR for aP2 and adiponectin expressions in 3T3-L1 cells treated with 20 lM harpagoside were performed as described in Fig. 3. Means ± SD from three independent experiments are shown (a, b, and c: p < 0.05 versus each other).
[29]. In parallel with these findings, PPAR-c agonists are reported to inhibit inflammatory cytokine production and increase plasma adiponectin level by transcriptional induction in adipose tissues [30]. In addition, it is known that TNF-a is one of the negative regulators of PPAR-c and adiponectin [31]. Our current findings suggest that harpagoside, which may act as a PPAR-c agonist, mitigated TNF-a-induced downregulation of PPAR-c and its target gene like adiponectin. In conclusion, harpagoside can attenuate TNF-a-stimulated increases in gene expression and secretion of adipokines in 3T3-L1 adipocytes. The effects of harpagoside were mediated by PPAR-c activation. The present study may contribute to the understanding of anti-inflammatory effects of harpagoside in adipocytes and provide a novel mechanism of harpagoside in preventing obesity-related pathologies. References [1] Tian J, Ye X, Shang Y, Deng Y, He K, Li X. Preparative isolation and purification of harpagoside and angroside C from the root of Scrophularia ningpoensis Hemsley by high-speed counter-current chromatography. J Sep Sci 2012;35:2659–64. [2] Lee MK, Choi OG, Park JH, Cho HJ, Ahn MJ, Kim SH, et al. Simultaneous determination of four active constituents in the roots of Scrophularia buergeriana by HPLC-DAD and LC-ESI-MS. J Sep Sci 2007;30:2345–50. [3] Qi J, Chen JJ, Cheng ZH, Zhou JH, Yu BY, Qiu SX. Iridoid glycosides from Harpagophytum procumbens D.C. (devil’s claw). Phytochemistry 2006;67:1372–7. [4] Georgiev MI, Ivanovska N, Alipieva K, Dimitrova P, Verpoorte R. Harpagoside: from Kalahari Desert to pharmacy shelf. Phytochemistry 2013;92:8–15. [5] Mncwangi N, Chen W, Vermaak I, Viljoen AM, Gericke N. Devil’s Claw-a review of the ethnobotany, phytochemistry and biological activity of Harpagophytum procumbens. J Ethnopharmacol 2012;143:755–71. [6] Chrubasik JE, Roufogalis BD, Chrubasik S. Evidence of effectiveness of herbal antiinflammatory drugs in the treatment of painful osteoarthritis and chronic low back pain. Phytother Res 2007;21:675–83.
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Please cite this article in press as: Kim TK, Park KS. Inhibitory effects of harpagoside on TNF-a-induced pro-inflammatory adipokine expression through PPAR-c activation in 3T3-L1 adipocytes. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.05.015
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Inhibitory effects of harpagoside on TNF-a-induced pro-inflammatory adipokine expression through PPAR-c activation in 3T3-L1 adipocytes Tae Kon Kim a, Kyoung Sik Park b,⇑ a b
Department of Medicinal Chemistry, College of Science and Engineering, Jungwon University, Republic of Korea Department of Biomedical Science, College of Science and Engineering, Cheongju University, Chungbuk, Republic of Korea
a r t i c l e
i n f o
Article history: Received 10 April 2015 Received in revised form 15 May 2015 Accepted 15 May 2015 Available online xxxx Keywords: Iridoid glycoside Herbal medicine Adipocyte Pro-inflammatory adipokine Atherosclerosis
a b s t r a c t Obesity is closely associated with increased production of pro-inflammatory adipokines, including interleukin (IL)-6, plasminogen activator inhibitor (PAI)-1, and adipose-tissue-derived monocyte chemoattractant protein (MCP)-1, which contribute to chronic and low-grade inflammation in adipose tissue. Harpagoside, a major iridoid glycoside present in devil’s claw, has been reported to show anti-inflammatory activities by suppression of lipopolysaccharide (LPS)-induced production of inflammatory cytokines in murine macrophages. The present study is aimed to investigate the effects of harpagoside on both tumor necrosis factor (TNF)-a-induced inflammatory adipokine expression and its underlying signaling pathways in differentiated 3T3-L1 cells. Harpagoside significantly inhibited TNF-a-induced mRNA synthesis and protein production of the atherogenic adipokines including IL-6, PAI-1, and MCP-1. Further investigation of the molecular mechanism revealed that pretreatment with harpagoside activated peroxisome proliferator–activated receptor (PPAR)-c. These findings suggest that the clinical application of medicinal plants which contain harpagoside may lead to a partial prevention of obesity-induced atherosclerosis by attenuating inflammatory responses. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Harpagoside is a naturally occurring iridoid glycoside found in many medicinal plants such as Scrophularia ningpoensis, Scrophularia buergeriana, and Harpagophytum procumbens [1–3]. These medicinal plants have been shown to exhibit a variety of biological activities and used as pharmaceutical products for the treatment of inflammatory ailment, rheumatoid arthritis, and osteoarthritis [4–6]. Harpagoside is believed to be a main bioactive compound related to the anti-inflammatory efficacy of these plants, and the harpagoside content is used to standardize commercial H. procumbens products, which should contain at least 1.2% of the compound according to the European Pharmacopoeia [7]. As for the molecular mechanism for anti-inflammatory activities of harpagoside, it was reported that harpagoside inhibits lipopolysaccharide (LPS)-induced production of inflammatory cytokines such as interleukin (IL)-1b, IL-6, and tumor necrosis factor (TNF)-a resulting from the both IjBa degradation and the
⇑ Corresponding author at: Department of Biomedical Science, College of Science and Engineering, Cheongju University, 298 Daesung-ro, Chungwon-gu, Cheongju-si, Chungbuk 363-764, Republic of Korea. Tel.: +82 43 229 8566; fax: +82 43 229 8559. E-mail address: [email protected] (K.S. Park).
nuclear translocation of NF-jB in RAW 264.7 cells [8]. However, the effects of harpagoside on obesity-related inflammatory responses in adipocytes have not been reported previously. Obesity, which is characterized by excessive accumulation of abdominal fat, is casually associated with the premature development of atherosclerosis, increased risk of stroke, and development of congestive heart failure [9]. Recent studies have indicated that the adipocyte secretes a variety of adipokines involved in energy metabolism, inflammation, and cardiovascular functions [10]. The cellular mechanisms linking obesity and atherosclerosis are complex and have not been fully elucidated. However, increasing evidences suggest that the changes of adipokines including IL-6, plasminogen activator inhibitor (PAI)-1, and monocyte chemoattractant protein (MCP)-1 due to excess adipose tissue may be a cause of atherosclerosis [11]. IL-6 is produced by many cell types and many tissues including adipose tissue. It is now well known that IL-6 production by adipose tissue is enhanced in obesity [12,13]. IL-6 has been recently proposed to play a central role in the link between obesity, inflammation, and coronary heart diseases [14]. PAI-1 is the primary inhibitor of plasminogen activation. Plasma levels of PAI-1 are markedly elevated in obese individuals as well as in patients with insulin resistance, type 2 diabetes, and cardiovascular diseases
http://dx.doi.org/10.1016/j.cyto.2015.05.015 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kim TK, Park KS. Inhibitory effects of harpagoside on TNF-a-induced pro-inflammatory adipokine expression through PPAR-c activation in 3T3-L1 adipocytes. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.05.015
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T.K. Kim, K.S. Park / Cytokine xxx (2015) xxx–xxx
(CVDs) [15,16]. PAI-1 is thought to be the link between obesity and increased risk for CVDs [17]. Although several tissues are known to produce PAI-1, adipose tissue appears to be the major contributor to elevated PAI-1 levels observed in cases of obesity [18,19]. Monocyte chemoattractant protein (MCP)-1 chemotactically recruits monocytes to sites of inflammation. Although this protein is traditionally thought to be expressed mainly endothelial cells and macrophages, it has been shown to be primarily expressed by adipose tissues [20,21]. Recently, MCP-1 has been reported to be a novel adipokine involved in the development of obesity-related insulin resistance and atherosclerosis [22]. The present study was designed to determine whether harpagoside attenuates TNF-a-induced secretion and mRNA production of the atherogenic adipokines including IL-6, PAI-1, and MCP-1 in differentiated 3T3-L1 adipocytes. In addition, the possible mechanisms for inhibitory effects of harpagoside on obesity-related inflammatory responses were examined.
2. Materials and methods
2.4. Evaluation of gene expression levels by quantitative real-time RTPCR For real-time RT-PCR, cDNA was prepared from 1 lg total RNA in a final reaction volume of 15 lL. Real-time RT-PCR was performed using 2 lL cDNA diluted with each of the gene-specific primer sets (Bioneer Inc., Korea). Each primer set was used at a concentration of 150 nM in a final reaction volume of 20 lL and reactions were performed on the Lightcycler 480 SYBR Green I Master (Roche Applied Science, Germany). The sequences for primers are: IL-6 (F: 50 -GCTACCAAACTGGATATAATCAGGA-30 and R: 50 -CCAGGTAGCTATGGTACTCCAGAA-30 ), PAI-1 (F: 50 -AGGATCGAG GTAAACGAGAGC-30 and R: 50 -GCGGGCTGAGATGACAAA-30 ), MCP-1 (F: 50 -GCCCCACTCACCTGCTGCTACT-30 and R: 50 -CCTGCTG CTGGTGATCCTCTTGT-30 ), aP2 (F: 50 -TGAAAGAAGTAGGAGTGGG C-30 and R: 50 -CTTCAGTCCAGGTCAACGTC-30 ), adiponectin (F: 50 -G TTCTACTGCAACATTCCGG-30 and R: 50 -TACACCTGGAGCCAGACTT G-30 ) and GAPDH (F: 50 -CGGTGCTGAGTATGTCTGTG-30 and R: 50 -G GTGGAGATGATGACCCTTT-30 ) [23,24]. All quantifications were performed in duplicate and the experiments were independently repeated three times.
2.1. Reagents Harpagoside with higher than 99.0% purity was purchased from Extrasynthese (Genay, France). Recombinant murine tumor necrosis factor (TNF)-a was from R&D Systems (Minneapolis, MN, USA). Insulin, dexamethasone, 3-isobutyl-1-methylxanthine, and GW9662 (2-chloro-5-nitrobenzanilide) were from Sigma (St. Louis, MO, USA). All tissue culture materials were from Gibco-BRL (Rockville, MD, USA). All the other chemicals used were purchased from Sigma (St. Louis, MO, USA). 2.2. 3T3-L1 cell culture and cytotoxicity test 3T3-L1 preadipocytes (American Type Culture Collection, Rockville, MD, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin–streptomycin (10,000 U/mL penicillin and 10,000 lg/mL streptomycin in 0.85% saline), and 1% (v/v) 100 lM pyruvate at 37 °C in 95% air 10% CO2. Differentiation of 2-day postconfluent preadipocytes (designated as day 0) was initiated with 0.5 mM 3-isobutyl-1-methylxanthine, 1 lM dexamethasone, and 1 lg/mL insulin in DMEM supplemented with 10% fetal bovine serum. After 48 h (day 2), the culture medium was replaced with DMEM supplemented with 10% fetal bovine serum and 1 lg/mL insulin, and the cells were then fed every other day with DMEM containing 10% fetal bovine serum. Harpagoside was reconstituted, filter sterilized, and stored at 20 °C. For each experiment, cells received harpagoside premixed with culture medium. TNF-a treatment was carried out after a 6-h pretreatment with harpagoside. Harpagoside was dissolved in dimethyl sulfoxide (DMSO) before it was added to culture medium. The final concentration of DMSO in culture medium was 0.1% (v/v). Possible cytotoxicity of harpagoside and TNF-a was assessed by the 3-(4,5-dimethylthiazo l-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) staining method. 2.3. Measurement of adipokines by ELISA The conditioned culture medium from 3T3-L1 adipocytes was collected from each sample. The concentrations of IL-6, PAI-1, and MCP-1 were assayed using a mouse IL-6 ELISA kit (Biosource Inc., Camarillo, CA, USA), a mouse PAI-1 ELISA kit (Molecular Innovations Inc., Southfield, MI, USA), and a mouse MCP-1 ELISA kit (R&D Systems, Minneapolis, MN, USA), respectively. All assays were performed according to the manufacturer’s instructions.
2.5. Luciferase reporter assay Luciferase assay was performed using the dual luciferase system (Promega, Madison, WI, USA) according to the manufacturer’s protocols. For PPAR-c luciferase assay, p4xUASg-tk-luc (a reporter plasmid), pRL-CMV (an internal control) and pM-PPAR-c (an expression plasmid for GAL4/PPAR-ligand binding domain chimera protein) were transfected into CV1 cells cultured on 24-well tissue culture plates. The transfection was performed by LipofectAMINE (Invitrogen, Carlsbad, CA, USA) using the manufacturer’s instructions. Twenty-four hours after the transfection, the transfected cells were cultured in medium containing harpagoside for an additional 24 h. 2.6. Statistics Data expressed as mean ± SD were obtained from three separate experiments. Statistical analysis of the data was performed by ANOVA and the unpaired t-test. P < 0.05 was considered to have significant difference. 3. Results 3.1. No cytotoxic effects of harpagoside and TNF-a on 3T3-L1 adipocytes We performed MTT test to evaluate the possibility of cytotoxicity of harpagoside and TNF-a on adipocytes that could misinterpret results. As shown in Fig. 1, harpagoside in concentrations of 10–100 lM with/without 10 ng/mL TNF-a did not show significant cytotoxicity. The relative viability of adipocytes was all greater than 95% when measured at 96 h after treatment. 3.2. Effects of harpagoside on the TNF-a-induced secretion of adipokines in 3T3-L1 adipocytes To examine the effects of harpagoside on TNF-a-induced secretion of adipokines, 3T3-L1 adipocytes were pretreated with various concentrations of harpagoside for 6 h and then incubated with 10 ng/mL TNF-a for 24 h. After incubation, conditioned medium was collected for ELISA assay. Treatment with harpagoside inhibited TNF-a-induced increase in the secretion of IL-6, PAI-1, and
Please cite this article in press as: Kim TK, Park KS. Inhibitory effects of harpagoside on TNF-a-induced pro-inflammatory adipokine expression through PPAR-c activation in 3T3-L1 adipocytes. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.05.015
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Fig. 1. No cytotoxicity of harpagoside and TNF-a to 3T3-L1 adipocytes. 3T3-L1 cells were incubated with increasing concentrations of harpagoside (from 10 to 100 lM, white bars) or with TNF-a (10 ng/mL, black bars) for 96 h. The cell viability was then determined by the MTT assay. Cell viability was normalized with that of untreated control (DMSO only treated) and is shown as percentages of relative cell viability. Values are mean ± SD of three independent experiments.
Fig. 2. Effects of harpagoside on TNF-a-stimulated adipokine production in 3T3-L1 adipocytes. 3T3-L1 adipocytes were exposed or not to increasing concentrations of harpagoside (5, 10, 20 and 50 lM) for 6 h and then were stimulated for 24 h by the addition of 10 ng/mL TNF-a. The secreted antigens of IL-6, PAI-1, and MCP-1 in conditioned medium were measured as described in Materials and Methods. Results represent the mean ± SD from three independent experiments. ##p < 0.01 versus untreated control; ⁄ p < 0.05, ⁄⁄p < 0.01 versus TNF-a-stimulated control.
Please cite this article in press as: Kim TK, Park KS. Inhibitory effects of harpagoside on TNF-a-induced pro-inflammatory adipokine expression through PPAR-c activation in 3T3-L1 adipocytes. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.05.015
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MCP-1 in dose-dependent manner with IC50 of 14.04 ± 2.60, 18.68 ± 0.48, and 12.18 ± 6.35 lM, respectively (Fig. 2). 3.3. Effects of harpagoside on the TNF-a-induced expression of adipokine genes in 3T3-L1 adipocytes To investigate whether harpagoside inhibited TNF-a-induced secretion of adipokines by altering the gene expression, real-time quantitative RT-PCR was carried out with total RNA extracted from 3T3-L1 adipocytes of each group. The gene expression of adipokines was measured after 3T3-L1 adipocytes were exposed to harpagoside for 6 h prior to TNF-a (10 ng/mL) treatment for 24 h. The enhanced production of IL-6, PAI-1, and MCP-1 mRNA by TNF-a was suppressed by harpagoside pretreatment (Fig. 3). At 20 lM of harpagoside, the mRNA production of IL-6, PAI-1, and MCP-1 was suppressed by 75.5%, 59.4%, and 69.5%, respectively. 3.4. Effects of harpagoside on the activity of PPAR-c To investigate the underlying mechanism of anti-inflammatory activities of harpagoside, we evaluated the effects of harpagoside on PPAR-c activity by using the luciferase ligand assay system. In assays with GAL4 and PPAR-c-chimera protein, harpagoside significantly activated GAL4/PPAR-c chimera transactivations in dose-dependent manner as shown in Fig. 4A. The level of PPAR-c activity with the treatment of 20 lM harpagoside was
approximately 2.0-fold higher than that in the vehicle control. Furthermore, to determine whether the effects of harpagoside are caused by PPAR-c activation, GW9662, a PPAR-c antagonist, was added together with harpagoside to the culture medium of 3T3-L1 adipocytes. GW9662 significantly increased both the mRNA production and protein synthesis of IL-6 and MCP-1 which had been reduced by the treatment of 20 lM harpagoside (Fig. 4B and C), indicating that PPAR-c plays an important role in the inhibition of the gene expression of the atherogenic adipokines by harpagoside. Moreover, to confirm the signaling pathway of harpagoside through PPAR-c activation, we examined the effects of harpagoside on the mRNA synthesis of PPAR-c target genes in TNF-a treated adipocytes. As shown in Fig. 5, TNF-a suppressed mRNA production of PPAR-c target genes including adipocyte fatty acid-binding protein (aP2) and adiponectin. Pretreatment of harpagoside attenuated TNF-a-mediated suppression of mRNA expressions of aP2 and adiponectin. These data indicate that harpagoside induces the expression of PPAR-c target genes through the PPAR-c-dependent pathway. 4. Discussions The anti-inflammatory activities of harpagoside have been mainly evaluated in mouse and rat carrageenan-induced oedema models [25,26]. Although the anti-inflammatory action of
Fig. 3. Effects of harpagoside on TNF-a-induced adipokine mRNA synthesis in 3T3-L1 adipocytes. After exposure to various concentrations of harpagoside (5, 10, 20 and 50 lM) for 6 h, 3T3-L1 adipocytes were stimulated with 10 ng/mL TNF-a for 24 h. Then cells were harvested, and total RNA was extracted for measuring the mRNA levels of IL-6, PAI-1, and MCP-1 by quantitative RT-PCR. Values are normalized to b-actin RNA expression levels and expressed relative to untreated control cells. Results represent the mean ± SD from three independent experiments. ##p < 0.01 versus untreated control; ⁄p < 0.05, ⁄⁄p < 0.01 versus TNF-a-stimulated control.
Please cite this article in press as: Kim TK, Park KS. Inhibitory effects of harpagoside on TNF-a-induced pro-inflammatory adipokine expression through PPAR-c activation in 3T3-L1 adipocytes. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.05.015
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Fig. 4. Effects of harpagoside on activation of PPAR-c. (A) Results of luciferase assays showing dose-dependencies of harpagoside on PPAR-c activities. p4xUASg-tk-luc and pRL-CMV were infected into CV1 cells together with pM-PPAR-c. Twenty-four hours after the infection, the cells were treated with harpagoside at various concentrations for 24 h. The activity of vehicle control was set at 100% and relative luciferase activity was presented as fold induction relative to that of the vehicle control. The values are mean ± SD of three independent experiments. ⁄⁄p < 0.01 compared with vehicle control. (B and C) Effects of PPAR-c inhibitor GW9662 on harpagoside-induced suppression of TNF-a-stimulated adipokine mRNA expression and protein synthesis in 3T3-L1 adipocytes. After exposure to 20 lM harpagoside in the absence or presence of GW9662 (10 lM) for 6 h and then were stimulated for 24 h by the addition of 10 ng/mL TNF-a. Then cells were harvested, and total RNA was extracted for measuring the mRNA levels of IL-6, PAI-1, and MCP-1 by quantitative RT-PCR. Values are normalized to b-actin RNA expression levels and expressed relative to untreated control cells (B). Moreover, the secreted antigens of IL-6, PAI-1, and MCP-1 in conditioned medium were measured as described in Materials and Methods (C). Results represent the mean ± SD from three independent experiments (a, b, and c: p < 0.05 versus each other).
harpagoside has been demonstrated in RAW 264.7 cells via NF-jB signaling pathway [8], the effects of harpagoside on obesity-related inflammatory responses in adipocytes have not been reported previously. The present study showed for the first time that harpagoside, a major component of H. procumbens, effectively suppressed the TNF-a-mediated increase in IL-6, PAI-1, and MCP-1 in 3T3-L1 adipocytes (Fig. 2). It was previously reported that the increased levels of TNF-a associated with obesity may significantly contribute to elevated plasma and adipose tissue expression levels of adipokines [27]. The TNF-a-induced changes in the mRNA expression of each adipokine were also inhibited by harpagoside (Fig. 3). These results indicate that harpagoside affects the TNF-a-mediated changes in the secretion of adipokines at the transcription level. In attempts to elucidate the possible mechanisms for inhibitory effects of harpagoside on obesity-related inflammatory responses,
it was found that harpagoside may act as an agonist for PPAR-c in luciferase ligand assays (Fig. 4A). This is confirmed by the fact that effects of harpagoside on regulation of TNF-a-induced adipokine protein production as well as mRNA expression disappeared by the addition of GW9662, a PPAR-c antagonist (Fig. 4B and C). In addition, harpagoside attenuated TNF-a-mediated suppression of mRNA expressions of PPAR-c target genes including aP2 and adiponectin, implying that harpagoside exerts the anti-inflammatory activities through the PPAR-c-dependent pathway (Fig. 5). Adiponectin expression is exclusive to adipose tissues and the mRNA expression level of adiponectin is low in obese mice and humans. Furthermore, a functional PPAR-responsive element (PPRE) has been identified in human adiponectin promoters [28]. PPAR-c promotes the transcription of adiponectin, which modulates glucose metabolism and exerts anti-inflammatory actions
Please cite this article in press as: Kim TK, Park KS. Inhibitory effects of harpagoside on TNF-a-induced pro-inflammatory adipokine expression through PPAR-c activation in 3T3-L1 adipocytes. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.05.015
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Fig. 5. Effects of harpagoside on TNF-a-induced mRNA synthesis of PPAR-c target genes in 3T3-L1 adipocytes. Real-time PCR for aP2 and adiponectin expressions in 3T3-L1 cells treated with 20 lM harpagoside were performed as described in Fig. 3. Means ± SD from three independent experiments are shown (a, b, and c: p < 0.05 versus each other).
[29]. In parallel with these findings, PPAR-c agonists are reported to inhibit inflammatory cytokine production and increase plasma adiponectin level by transcriptional induction in adipose tissues [30]. In addition, it is known that TNF-a is one of the negative regulators of PPAR-c and adiponectin [31]. Our current findings suggest that harpagoside, which may act as a PPAR-c agonist, mitigated TNF-a-induced downregulation of PPAR-c and its target gene like adiponectin. In conclusion, harpagoside can attenuate TNF-a-stimulated increases in gene expression and secretion of adipokines in 3T3-L1 adipocytes. The effects of harpagoside were mediated by PPAR-c activation. The present study may contribute to the understanding of anti-inflammatory effects of harpagoside in adipocytes and provide a novel mechanism of harpagoside in preventing obesity-related pathologies. References [1] Tian J, Ye X, Shang Y, Deng Y, He K, Li X. Preparative isolation and purification of harpagoside and angroside C from the root of Scrophularia ningpoensis Hemsley by high-speed counter-current chromatography. J Sep Sci 2012;35:2659–64. [2] Lee MK, Choi OG, Park JH, Cho HJ, Ahn MJ, Kim SH, et al. Simultaneous determination of four active constituents in the roots of Scrophularia buergeriana by HPLC-DAD and LC-ESI-MS. J Sep Sci 2007;30:2345–50. [3] Qi J, Chen JJ, Cheng ZH, Zhou JH, Yu BY, Qiu SX. Iridoid glycosides from Harpagophytum procumbens D.C. (devil’s claw). Phytochemistry 2006;67:1372–7. [4] Georgiev MI, Ivanovska N, Alipieva K, Dimitrova P, Verpoorte R. Harpagoside: from Kalahari Desert to pharmacy shelf. Phytochemistry 2013;92:8–15. [5] Mncwangi N, Chen W, Vermaak I, Viljoen AM, Gericke N. Devil’s Claw-a review of the ethnobotany, phytochemistry and biological activity of Harpagophytum procumbens. J Ethnopharmacol 2012;143:755–71. [6] Chrubasik JE, Roufogalis BD, Chrubasik S. Evidence of effectiveness of herbal antiinflammatory drugs in the treatment of painful osteoarthritis and chronic low back pain. Phytother Res 2007;21:675–83.
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Please cite this article in press as: Kim TK, Park KS. Inhibitory effects of harpagoside on TNF-a-induced pro-inflammatory adipokine expression through PPAR-c activation in 3T3-L1 adipocytes. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.05.015