http://informahealthcare.com/ipi ISSN: 0892-3973 (print), 1532-2513 (electronic) Immunopharmacol Immunotoxicol, 2015; 37(2): 185–192 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/08923973.2015.1009997
RESEARCH ARTICLE
Osthole inhibits inflammatory cytokine release through PPARa/c-mediated mechanisms in LPS-stimulated 3T3-L1 adipocytes Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Kainan University on 04/03/15 For personal use only.
Xiao-li Wang1*, Xiang Shang1*, Yan Cui1, Xi Zhao2, Yan Zhang1, and Mei-lin Xie1 1
Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu Province, China and 2Department of Pharmaceutical Laboratory, Medical College of Nantong University, Nantong, Jiangsu Province, China Abstract
Keywords
Context: Peroxisome proliferator-activated receptor (PPAR) a/g may control inflammatory response by regulating the nuclear factor-kappa B (NF-kB). Osthole may be a dual agonist of PPARa/g, but whether or not osthole may inhibit inflammatory cytokines in cultured 3T3-L1 adipocytes is unclear. Objective: We investigated the action of osthole and its potential mechanisms in lipopolysaccharide (LPS)-stimulated 3T3-L1 adipocytes. Materials and methods: The 3T3-L1 adipocytes stimulated with LPS were cultured and treated with different concentrations of osthole. The inflammatory cytokines including tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) in cultured supernatants were detected by the enzymelinked immunosorbent assay (ELISA) method, and the protein expressions of PPARa/g and NFkB p65 in adipocytes were detected by the Western blot method, respectively. Results: Following treatment of adipocytes with osthole 0.1–1.6 mM, the TNF-a and IL-6 levels in cultured supernatants were decreased, and the NF-kB p65 protein expression in adipocytes was also decreased, while the PPARa/g protein expressions were increased. After pretreatment of adipocytes with specific inhibitor(s) of PPARa and /or PPARg, the inhibitory effects of osthole on TNF-a and IL-6 were decreased or almost cancelled, and the effects on NF-kB p65 protein expression also exhibited similar variations. Conclusion: Osthole could inhibit the TNF-a and IL-6 production in LPS-stimulated adipocytes, and its mechanism might be related to reduction of NF-kB expression via activation of PPARa/g.
IL-6, osthole, PPARa/g, TNF-a, 3T3-L1 adipocytes
Introduction Adipose tissue has long been acted as an energy storage organ, but it is now recognized as an active endocrine organ1 and can secrete a series of adipocytokines that are known to involve in the development of numerous obesity-related diseases, such as nonalcoholic steatohepatitis (NASH)2. The tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6), two inflammatory cytokines expressed and released by adipose tissue, play the important roles in the process of obesityinduced inflammation and lipid metabolic disorders3–5. NASH is characterized by hepatic steatosis and inflammation, and this disease can progress to liver fibrosis, cirrhosis, and ultimately hepatocellular carcinoma6. Although the pathogenesis of NASH is not yet clear completely, the ‘‘two hits’’ theory proposed by Day and James is widely accepted7. The TNF-a and IL-6 originated from adipose tissue are considered *These authors contributed equally to this work. Address for correspondence: Prof. Mei-lin Xie, Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, 199 Renai Road, Suzhou Industrial Park, Suzhou 215123, Jiangsu Province, China. Tel: +86 512 69566553. Fax: +86 512 65882089. E-mail: [email protected]
History Received 5 August 2014 Revised 30 December 2014 Accepted 16 January 2015 Published online 18 February 2015
significant to NASH through interference with the process of two hits2,5. Peroxisome proliferator-activated receptor (PPAR) a/g are nuclear receptors that are known to control the lipid metabolism, adipocyte differentiation, and inflammatory response8–10. It is reported that the anti-inflammatory effects of glucocorticoids, a representative of anti-inflammatory drugs, are associated with PPARa activation11, and PPARg agonists are more specific for controlling the inflammatory response12. Recent studies have also indicated that PPARa/g have a central role in the generation and development of NASH and may be the potential therapeutic targets9,10,13. Activation of PPARa/g can ameliorate inflammatory response by inhibition of nuclear factor-kappa B (NF-kB) and subsequent reduction of inflammatory cytokine production14,15 including TNF-a and IL-6. The latter may alleviate the ‘‘second hit’’ itself and insulin resistance from the ‘‘first hit’’16. Osthole is an active component isolated from the fruit of Cnidium monnieri (L.) Cusson, one of the Chinese herbal medicines. Modern pharmacological researches have proven that osthole has many functions and shows anti-osteoporosis17, anti-inflammation18, anti-cardiac hypertrophy19, anti-insulin resistance20, and inhibition of hepatic
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oxidative stress21. Recently, our studies found that osthole possessed a therapeutic effect on high-fat and high-sucroseinduced steatohepatitis in rats, inhibited fatty acid synthesis and release in cultured 3T3-L1 adipocytes, and simultaneously upregulated the PPAR / gene expressions in liver and cultured adipocytes22,23. These findings suggest that osthole may exert a beneficial effect on NASH. In the present study, we further investigated whether osthole might inhibit inflammatory cytokine release from lipopolysaccharide (LPS)-stimulated 3T3-L1 adipocytes via PPARa/g-mediated pathways, which may be contributory to amelioration of NASH.
Measurement of cell viability
Materials and methods
Experimental design
Reagents
The 3T3-L1 adipocytes were seeded in 24-well culture plates at a destiny of 1 105 cells/ml and were divided into five groups as follows: H-DMEM (control), H-DMEM with 1% DMSO (vehicle), and osthole 0.1, 0.4, and 1.6 mM groups. In the osthole-treated groups, the cells were incubated with different concentrations of osthole for 8 h. For the other study, the cells were seeded in 6-well culture plates, and the LPSstimulated group (model) was added additionally. The osthole-treated cells were stimulated with LPS (a final concentration was 2 mg/ml) for 6 h after pretreatment with osthole for 2 h. To determine whether the effect of osthole on inflammatory cytokine release was associated with the PPARa/g pathway, the cultured cells were pretreated with 4 mM MK886 (a specific PPARa inhibitor) and/or 10 mM GW9662 (a specific PPARg inhibitor) for 2 h before incubation with osthole 1.6 mM for 8 h. The cells were divided into following groups, namely H-DMEM (control), H-DMEM with 1% DMSO (vehicle), LPS 2 mg/ml (model), osthole 1.6 mM, LPS plus osthole 1.6 mM, MK886 (4 mM) and/or GW9662 (10 mM), LPS puls MK886 (4 mM) and/or GW9662 (10 mM), LPS plus MK886 (4 mM) and/or GW9662 (10 mM) plus osthole 1.6 mM. Finally, the cells were stimulated with LPS 2 mg/ml for 6 h. These cultured supernatants and cells were then collected for measurement of TNF-a and IL-6 levels as well as PPARa/g and NF-kB p65 protein expressions, respectively.
Osthole was kindly provided by Dr. Jia Zhou of Green Fount Natural Product Co., Ltd. (Xi’an, China) and solubilized in 1% dimethyl sulfoxide (DMSO) solution, and the purity was 98% as determined by high-performance liquid chromatography. Dexamethasone, insulin, 3-isobutyl1-methylxanthine, DMSO, LPS, and 3-(4,5-dimethylthiazoyl2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). High/low glucose Dulbecco’s modified Eagles medium (H/L-DMEM) and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT). Newborn calf serum was the product of Gibco (Grand Island, NY). The assay kits for TNF-a and IL-6 were provided by Shanghai Westang Bio-Tech Co., Ltd. (Shanghai, China). MK886 (a specific PPARa inhibitor) was the product of Cayman Chemical Company (Ann Arbor, MI). GW9662 (a specific PPARg inhibitor) was the product of Alexis Biochemicals Corporation (Lausen, Switzerland). AntiPPARa antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PPARg, anti-NF-kB p65, and antib-actin antibodies were obtained from CST Company (Boston, MA). Differentiation of 3T3-L1 preadipocytes Mouse 3T3-L1 preadipocytes, purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China), were cultured in L-DMEM solution supplemented with 10% calf serum and maintained at 37 C in a humidified atmosphere with 5% CO2 in 6-well culture plates. After cells were completely confluent, the differentiation of cells was induced with 0.25 mM 3-isobutyl-1-methylxanthine, 0.5 mM dexamethasone, and 1 mg/ml insulin in H-DMEM solution supplemented with 10% FBS. Three days later, the medium was replaced with H-DMEM solution containing 1 mg/ml insulin and 10% FBS for 2 d. Finally, the cells were incubated with H-DMEM solution containing 10% FBS for another 8 d. The culture medium was changed every 2 d. After 13 d of differentiation, the cells were stained with Oil Red O solution and examined under a light microscope to evaluate the degree of differentiation. About 90% of the preadipocytes might translate into mature adipocytes, characterized by a circular-oval shape with fat deposits in small vacuoles.
The 3T3-L1 adipocytes were seeded in 96-well culture plates at a density of 1 104 cells/well and incubated with osthole at 0.1, 0.4, 1.6, and 6.4 mM for 3, 6, 12, 24, and 48 h, respectively. After medium removal, the cells were incubated with MTT solution (5 mg/ml in phosphate-buffered solution (137 mM NaCl, 2.68 mM KCl, 1.47 mM KH2PO4, and 8.1 mM Na2HPO4, pH 7.4) for 4 h and the resulting formazan was solubilized with DMSO (150 ml). The absorption was measured at 570 nm in an enzyme-linked immunosorbent assay reader. The absorbance of control cells (untreated) was considered as 100% cell viability.
Enzyme-linked immunosorbent assay (ELISA) The TNF-a and IL-6 levels in cultured supernatants were determined by the ELISA method according to the instructions of the manufacturer on a VersaMax plate reader (Molecular Devices, Sunnyvale, CA). Western blot assay An aliquot of 20–40 mg of protein from each sample was loaded onto 10% SDS-polyacrylamide gel and subjected to electrophoresis under constant current, and subsequently transferred to nitrocellulose membranes (Millipore, Billerica, MA). The membrane was closed with 5% skimmed milk in Tris-buffered solution with 0.1% Tween 20 for 2 h at room temperature and then incubated with the primary antibody of PPARa (1:800 dilution) or PPARg (1:200 dilution) or NF-kB p65 (1:1000 dilution) or b-actin (1:2000 dilution) at 4 C overnight. Afterwards, the membrane was washed and
DOI: 10.3109/08923973.2015.1009997
Osthole inhibits inflammatory cytokine release through PPAR / -mediated mechanisms
incubated with fluorescent secondary antibody for 1 h at room temperature. The ratio of the protein interested was subjected to b-actin and was densitometrically analyzed by the Odyssey infrared imaging system. Statistical analysis
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Data are expressed as mean ± SD of at least three independent experiments. The one-way ANOVA followed by a post hoc LSD test was used for comparisons between groups. The statistical analysis was conducted using the SPSS 18.0 program (SPSS Inc., Chicago, IL), and differences were considered at a value of p50.0 5.
Results Effect of osthole on cell viability The goal of this study was to investigate the effect of osthole on the inflammatory cytokine release in cultured 3T3-L1 adipocytes, the dosage of osthole should not affect cell viability. Therefore, we examined the cytotoxicity of osthole by MTT assay. The results revealed that the cell survival rate did not differ obviously when 3T3-L1 adipocytes were treated with osthole 0.1, 0.4, 1.6, and 6.4 mM for 3, 6, 12, 24, and 48 h, respectively (Figure 1), indicating that osthole, in concentrations from 0.1 to 6.4 mM, had no cytotoxicity on 3T3-L1 adipocytes, and the concentrations of osthole might be used in the experiments. Effects of osthole on TNF-a and IL-6 levels in cultured supernatants of 3T3-L1 adipocytes To determine whether osthole might affect the inflammatory cytokine production in normal adipocytes, the TNF-a and
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IL-6 levels in cultured supernatants were measured by the ELISA method. The experimental results showed that the TNF-a and IL-6 levels between control and vehicle groups were not significantly different (Figure 2), indicating that 1% DMSO did not affect the synthesis and release of these cytokines in cultured adipocytes. Following treatment of adipocytes with osthole 0.1–1.6 mM for 8 h, the TNF-a and IL-6 levels in cultured supernatants also exhibited no obvious changes as compared with the vehicle group (Figure 2), suggesting that osthole treatment might not alter the background levels of TNF-a and IL-6 in normal adipocytes under current treatment conditions. Effects of osthole on TNF-a and IL-6 levels in cultured supernatants of 3T3-L1 adipocytes stimulated with LPS LPS may potently induce adipocyte activation and the production of inflammatory cytokines. To examine the effects of osthole on the synthesis and release of TNF-a and IL-6 in activated adipocytes, LPS was used in the experiments. The present results showed that after stimulation with LPS 2 mg/ml for 6 h, the levels of TNF-a and IL-6 in cultured supernatants were significantly increased as compared with the vehicle group (p50.05, Figure 3). The addition of osthole could prevent the increments of TNF-a and IL-6 levels. As shown in Figure 3, the TNF-a level was suppressed by 6.5% at 0.1 mM, 20.8% at 0.4 mM, and 56.0% at 1.6 mM, and the effect showed a good dose–effect relationship. Also, the IL-6 level was inhibited by 44.4% at 0.1 mM, 38.3% at 0.4 mM, and 28.0% at 1.6 mM osthole, but the dose–effect relationship was not observed, the most effective concentration of osthole was 0.1 mM (p50.01, Figure 3).
Figure 1. Cell viability after treatment with osthole 0.1–6.4 mM for 3–48 h. Data are presented as mean ± SD, with n ¼ 4 per group.
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Figure 2. The TNF-a and IL-6 levels in cultured supernatants after treatment of 3T3-L1 adipocytes with osthole for 8 h. Data are presented as mean ± SD, with n ¼ 4 per group.
Figure 3. The TNF-a and IL-6 levels in cultured supernatants of 3T3-L1 adipocytes stimulated with LPS 2 mg/ml for 6 h after pretreatment with osthole for 2 h. Data are presented as mean ± SD, with n ¼ 4 per group. *p50.05 versus the vehicle group; #p50.05, ##p50.01 versus the LPS 2 mg/ml group.
Effects of osthole on PPARa/c and NF-kB p65 protein expressions in 3T3-L1 adipocytes stimulated with LPS To investigate whether the anti-inflammatory effect of osthole was associated with PPARa/g-NF-kB pathways, the protein levels of PPARa/g and NF-kB expressions in adipocytes were determined by Western blot assay. The results are shown in Figure 4. Compared with the vehicle group, the protein expressions of PPARa and NF-kB p65 in 3T3-L1 adipocytes in the LPS-stimulated group was low and high (p50.01), but the protein expression of PPARg was unchanged. The addition of 0.1–1.6 mM osthole could dose dependently increase the PPARa/g protein expressions
(Figure 4a and b) and decrease the NF-kB p65 protein expression (Figure 4c) in 3T3-L1 cells stimulated with LPS (p50.05 or p50.01). Effects of PPAR a/c inhibitors on osthole-reduced TNF-a and IL-6 levels in cultured supernatants of 3T3-L1 adipocytes stimulated with LPS In order to further confirm whether osthole reduced TNF-a and IL-6 production via PPARa/g pathways, a PPARa inhibitor MK886 (4 mM) and a PPARg inhibitor GW9662 (10 mM) were applied. After pretreatment of adipocytes with MK886 for 2 h, the inhibitory effects of osthole on TNF-a and
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DOI: 10.3109/08923973.2015.1009997
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Figure 4. The PPARa/g and NF-kB p65 protein expressions in 3T3-L1 adipocytes stimulated with LPS 2 mg/ml for 6 h after pretreatment with osthole for 2 h. Data are presented as mean ± SD, with n ¼ 3 per group. **p50.01 versus the vehicle group; #p50.05, ##p50.01 versus the LPS 2 mg/ml group.
IL-6 were attenuated (p50.05, Figure 5a), but not completely halted (p50.05, Figure 5a). Likewise, after pretreatment of adipocytes with GW9662 or MK886 plus GW9662 for 2 h, the inhibitory effects of osthole on TNF-a and IL-6 were notably alleviated or almost cancelled (p50.05, Figure 5b and c). The effects of MK886 and/or GW9662 per se on the two inflammatory cytokines were not found. Effects of PPAR a/c inhibitors on osthole-reduced NF-iB p65 protein expression in 3T3-L1 adipocytes stimulated with LPS NF-kB is known to be required for the induction of inflammatory cytokines, PPAR a/g may affect its expression. The present experimental results showed that after pretreatment of 3T3-L1 adipocytes with PPAR a inhibitor MK886 for 2 h, the osthole-reduced protein expression of NF-kB p65 was attenuated (p50.05, Figure 6a), but not completely halted (p50.05, Figure 6a). After the cells were pretreated with PPAR g inhibitor GW9662 or MK886 plus GW9662 for 2 h, the inhibitory effect of osthole on NF-kB p65 protein expression was almost cancelled (p50.05, Figure 6b and c). The effects of MK886 and/or GW9662 per se on NF-kB p65 protein expression were not found.
Discussion Recent literature data show that circulating inflammatory cytokines are elevated in patients with NASH24,25, and the origination from adipose tissue may contribute about 30%26. These inflammatory cytokines not only induce the ‘‘second hits’’ of liver but also enhance the hepatic lipid accumulation via induction of insulin resistance27–29. Therefore, the reduction of inflammatory cytokines in adipose tissue is very important to amelioration of NASH. Our present results showed that osthole could decrease the TNF-a and IL-6 levels in cultured supernatants of 3T3-L1 adipocytes stimulated with LPS, suggesting that the therapeutic effect of osthole on
NASH might be partly attributable to its inhibitory effect on TNF-a and IL-6 synthesis and release in adipose tissue. However, we found that the inhibitory effect of osthole on IL-6 was decreased with increasing concentrations of osthole. The potential reason might be the enhancement of ERK1/2 phosphorylation by osthole30, which might result in the increment of IL-6 release. Several studies in the experimental models indicate that activation of PPARa/g can inhibit the generation of inflammatory cytokines through interference with the inflammatory transcription factor NF-kB14,31. In order to explore the possible mechanisms of osthole in inhibiting TNF-a and IL-6 production, we examined the effects on PPARa/g and NF-kB p65 protein expressions in cultured adipocytes. The present results, in accordance with the results of mRNA expressions23, showed that osthole could increase the PPARa/g protein expressions in a dose-dependent manner. Accordingly, the NF-kB p65 protein expression was decreased, and the finding was also consistent with the result of in vitro cultured macrophages30. So, we thought that inhibitory effects of osthole on TNF-a and IL-6 might be related to reduction of NF-kB inflammatory signaling pathway via activation of PPARa/g. But it is necessary to investigate the exact effect of osthole on the modulation of NF-kB. If so, how osthole affects the nuclear translocation or its activity is a valuable issue to research further. In order to further verify whether osthole suppressed the TNF-a and IL-6 release from LPS-stimulated adipocytes through PPARa/g-NF-kB pathways, a specific PPARa inhibitor MK886 and a specific PPARg inhibitor GW9662 were used32,33. The results showed that after pretreatment of adipocytes with MK886 and/or GW9662 for 2 h, the inhibitory effects of osthole on TNF-a and IL-6 were decreased or almost cancelled, and the effects on NF-kB p65 protein expression also exhibited similar variations. These results revealed that PPARa/g-NF-kB signaling pathways involved in the inhibitory effects of osthole on TNF-a and IL-6 production in adipocytes. But our experimental results disclosed that
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Figure 5. Effects of osthole on TNF-a and IL-6 levels in cultured supernatants of 3T3-L1 adipocytes stimulated with LPS after the cells were pretreated with PPARa inhibitor MK886 4 mM (a), PPARg inhibitor GW9662 10 mM (b), and PPARa inhibitor MK886 4 mM plus PPARg inhibitor GW9662 10 mM (c) for 2 h. Data are presented as mean ± SD, with n ¼ 3 per group. *p50.05, **p50.01 versus the vehicle group; #p50.05, ##p50.01 versus the LPS 2 mg/ml group; &p50.05, &&p50.01 versus the GW9662 and/or MK886 group; 4p50.05 versus the osthole 1.6 mM + LPS 2 mg/ml group; mp50.05 versus the MK886 4 mM + LPS 2 mg/ml group.
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DOI: 10.3109/08923973.2015.1009997
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Figure 6. Effect of osthole on NF-kB p65 protein expression in 3T3-L1 adipocytes stimulated with LPS after the cells were pretreated with PPARa inhibitor MK886 4 mM (a), PPARg inhibitor GW9662 10 mM (b), and PPARa inhibitor MK886 4 mM plus PPARg inhibitor GW9662 10 mM (c) for 2 h. Data are presented as mean ± SD, with n ¼ 3 per group. **p50.01 versus the vehicle group; #p50.05 versus the LPS 2 mg/ml group; &&p50.01 versus the GW9662 and/or MK886 group; 4p50.05 versus the osthole 1.6 mM + LPS 2 mg/ml group; mp50.05 versus the MK886 4 mM + LPS 2 mg/ml group.
the pretreatment with 4 mM MK886 did not completely cancelled the inhibitory effects of osthole on the inflammatory cytokine release and NF-kB protein expression, suggesting that the concentration of MK886 used in this study might not be high enough and the osthole still worked under the conditions. In summary, our results further demonstrated that osthole could inhibit the TNF-a and IL-6 production in adipocytes, and the inhibitory effects might be related to reduction of NF-kB expression via activation of PPARa/g. This may be a novel pharmacological effect of osthole in the reduction of inflammatory response and insulin resistance, and be good for the prevention and treatment of NASH.
Declaration of interest This research was sponsored by the National Natural Science Foundation of China (No. 81173067), the Postgraduate Innovative Foundation of Jiangsu Province (No. CXLZ120854), the Excellent Postgraduate Items of Soochow University, and the Priority Academic Program Development of Jiangsu Higher Education Institutions, China. The authors declare no conflicts of interest.
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RESEARCH ARTICLE
Osthole inhibits inflammatory cytokine release through PPARa/c-mediated mechanisms in LPS-stimulated 3T3-L1 adipocytes Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Kainan University on 04/03/15 For personal use only.
Xiao-li Wang1*, Xiang Shang1*, Yan Cui1, Xi Zhao2, Yan Zhang1, and Mei-lin Xie1 1
Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu Province, China and 2Department of Pharmaceutical Laboratory, Medical College of Nantong University, Nantong, Jiangsu Province, China Abstract
Keywords
Context: Peroxisome proliferator-activated receptor (PPAR) a/g may control inflammatory response by regulating the nuclear factor-kappa B (NF-kB). Osthole may be a dual agonist of PPARa/g, but whether or not osthole may inhibit inflammatory cytokines in cultured 3T3-L1 adipocytes is unclear. Objective: We investigated the action of osthole and its potential mechanisms in lipopolysaccharide (LPS)-stimulated 3T3-L1 adipocytes. Materials and methods: The 3T3-L1 adipocytes stimulated with LPS were cultured and treated with different concentrations of osthole. The inflammatory cytokines including tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) in cultured supernatants were detected by the enzymelinked immunosorbent assay (ELISA) method, and the protein expressions of PPARa/g and NFkB p65 in adipocytes were detected by the Western blot method, respectively. Results: Following treatment of adipocytes with osthole 0.1–1.6 mM, the TNF-a and IL-6 levels in cultured supernatants were decreased, and the NF-kB p65 protein expression in adipocytes was also decreased, while the PPARa/g protein expressions were increased. After pretreatment of adipocytes with specific inhibitor(s) of PPARa and /or PPARg, the inhibitory effects of osthole on TNF-a and IL-6 were decreased or almost cancelled, and the effects on NF-kB p65 protein expression also exhibited similar variations. Conclusion: Osthole could inhibit the TNF-a and IL-6 production in LPS-stimulated adipocytes, and its mechanism might be related to reduction of NF-kB expression via activation of PPARa/g.
IL-6, osthole, PPARa/g, TNF-a, 3T3-L1 adipocytes
Introduction Adipose tissue has long been acted as an energy storage organ, but it is now recognized as an active endocrine organ1 and can secrete a series of adipocytokines that are known to involve in the development of numerous obesity-related diseases, such as nonalcoholic steatohepatitis (NASH)2. The tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6), two inflammatory cytokines expressed and released by adipose tissue, play the important roles in the process of obesityinduced inflammation and lipid metabolic disorders3–5. NASH is characterized by hepatic steatosis and inflammation, and this disease can progress to liver fibrosis, cirrhosis, and ultimately hepatocellular carcinoma6. Although the pathogenesis of NASH is not yet clear completely, the ‘‘two hits’’ theory proposed by Day and James is widely accepted7. The TNF-a and IL-6 originated from adipose tissue are considered *These authors contributed equally to this work. Address for correspondence: Prof. Mei-lin Xie, Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, 199 Renai Road, Suzhou Industrial Park, Suzhou 215123, Jiangsu Province, China. Tel: +86 512 69566553. Fax: +86 512 65882089. E-mail: [email protected]
History Received 5 August 2014 Revised 30 December 2014 Accepted 16 January 2015 Published online 18 February 2015
significant to NASH through interference with the process of two hits2,5. Peroxisome proliferator-activated receptor (PPAR) a/g are nuclear receptors that are known to control the lipid metabolism, adipocyte differentiation, and inflammatory response8–10. It is reported that the anti-inflammatory effects of glucocorticoids, a representative of anti-inflammatory drugs, are associated with PPARa activation11, and PPARg agonists are more specific for controlling the inflammatory response12. Recent studies have also indicated that PPARa/g have a central role in the generation and development of NASH and may be the potential therapeutic targets9,10,13. Activation of PPARa/g can ameliorate inflammatory response by inhibition of nuclear factor-kappa B (NF-kB) and subsequent reduction of inflammatory cytokine production14,15 including TNF-a and IL-6. The latter may alleviate the ‘‘second hit’’ itself and insulin resistance from the ‘‘first hit’’16. Osthole is an active component isolated from the fruit of Cnidium monnieri (L.) Cusson, one of the Chinese herbal medicines. Modern pharmacological researches have proven that osthole has many functions and shows anti-osteoporosis17, anti-inflammation18, anti-cardiac hypertrophy19, anti-insulin resistance20, and inhibition of hepatic
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oxidative stress21. Recently, our studies found that osthole possessed a therapeutic effect on high-fat and high-sucroseinduced steatohepatitis in rats, inhibited fatty acid synthesis and release in cultured 3T3-L1 adipocytes, and simultaneously upregulated the PPAR / gene expressions in liver and cultured adipocytes22,23. These findings suggest that osthole may exert a beneficial effect on NASH. In the present study, we further investigated whether osthole might inhibit inflammatory cytokine release from lipopolysaccharide (LPS)-stimulated 3T3-L1 adipocytes via PPARa/g-mediated pathways, which may be contributory to amelioration of NASH.
Measurement of cell viability
Materials and methods
Experimental design
Reagents
The 3T3-L1 adipocytes were seeded in 24-well culture plates at a destiny of 1 105 cells/ml and were divided into five groups as follows: H-DMEM (control), H-DMEM with 1% DMSO (vehicle), and osthole 0.1, 0.4, and 1.6 mM groups. In the osthole-treated groups, the cells were incubated with different concentrations of osthole for 8 h. For the other study, the cells were seeded in 6-well culture plates, and the LPSstimulated group (model) was added additionally. The osthole-treated cells were stimulated with LPS (a final concentration was 2 mg/ml) for 6 h after pretreatment with osthole for 2 h. To determine whether the effect of osthole on inflammatory cytokine release was associated with the PPARa/g pathway, the cultured cells were pretreated with 4 mM MK886 (a specific PPARa inhibitor) and/or 10 mM GW9662 (a specific PPARg inhibitor) for 2 h before incubation with osthole 1.6 mM for 8 h. The cells were divided into following groups, namely H-DMEM (control), H-DMEM with 1% DMSO (vehicle), LPS 2 mg/ml (model), osthole 1.6 mM, LPS plus osthole 1.6 mM, MK886 (4 mM) and/or GW9662 (10 mM), LPS puls MK886 (4 mM) and/or GW9662 (10 mM), LPS plus MK886 (4 mM) and/or GW9662 (10 mM) plus osthole 1.6 mM. Finally, the cells were stimulated with LPS 2 mg/ml for 6 h. These cultured supernatants and cells were then collected for measurement of TNF-a and IL-6 levels as well as PPARa/g and NF-kB p65 protein expressions, respectively.
Osthole was kindly provided by Dr. Jia Zhou of Green Fount Natural Product Co., Ltd. (Xi’an, China) and solubilized in 1% dimethyl sulfoxide (DMSO) solution, and the purity was 98% as determined by high-performance liquid chromatography. Dexamethasone, insulin, 3-isobutyl1-methylxanthine, DMSO, LPS, and 3-(4,5-dimethylthiazoyl2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). High/low glucose Dulbecco’s modified Eagles medium (H/L-DMEM) and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT). Newborn calf serum was the product of Gibco (Grand Island, NY). The assay kits for TNF-a and IL-6 were provided by Shanghai Westang Bio-Tech Co., Ltd. (Shanghai, China). MK886 (a specific PPARa inhibitor) was the product of Cayman Chemical Company (Ann Arbor, MI). GW9662 (a specific PPARg inhibitor) was the product of Alexis Biochemicals Corporation (Lausen, Switzerland). AntiPPARa antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PPARg, anti-NF-kB p65, and antib-actin antibodies were obtained from CST Company (Boston, MA). Differentiation of 3T3-L1 preadipocytes Mouse 3T3-L1 preadipocytes, purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China), were cultured in L-DMEM solution supplemented with 10% calf serum and maintained at 37 C in a humidified atmosphere with 5% CO2 in 6-well culture plates. After cells were completely confluent, the differentiation of cells was induced with 0.25 mM 3-isobutyl-1-methylxanthine, 0.5 mM dexamethasone, and 1 mg/ml insulin in H-DMEM solution supplemented with 10% FBS. Three days later, the medium was replaced with H-DMEM solution containing 1 mg/ml insulin and 10% FBS for 2 d. Finally, the cells were incubated with H-DMEM solution containing 10% FBS for another 8 d. The culture medium was changed every 2 d. After 13 d of differentiation, the cells were stained with Oil Red O solution and examined under a light microscope to evaluate the degree of differentiation. About 90% of the preadipocytes might translate into mature adipocytes, characterized by a circular-oval shape with fat deposits in small vacuoles.
The 3T3-L1 adipocytes were seeded in 96-well culture plates at a density of 1 104 cells/well and incubated with osthole at 0.1, 0.4, 1.6, and 6.4 mM for 3, 6, 12, 24, and 48 h, respectively. After medium removal, the cells were incubated with MTT solution (5 mg/ml in phosphate-buffered solution (137 mM NaCl, 2.68 mM KCl, 1.47 mM KH2PO4, and 8.1 mM Na2HPO4, pH 7.4) for 4 h and the resulting formazan was solubilized with DMSO (150 ml). The absorption was measured at 570 nm in an enzyme-linked immunosorbent assay reader. The absorbance of control cells (untreated) was considered as 100% cell viability.
Enzyme-linked immunosorbent assay (ELISA) The TNF-a and IL-6 levels in cultured supernatants were determined by the ELISA method according to the instructions of the manufacturer on a VersaMax plate reader (Molecular Devices, Sunnyvale, CA). Western blot assay An aliquot of 20–40 mg of protein from each sample was loaded onto 10% SDS-polyacrylamide gel and subjected to electrophoresis under constant current, and subsequently transferred to nitrocellulose membranes (Millipore, Billerica, MA). The membrane was closed with 5% skimmed milk in Tris-buffered solution with 0.1% Tween 20 for 2 h at room temperature and then incubated with the primary antibody of PPARa (1:800 dilution) or PPARg (1:200 dilution) or NF-kB p65 (1:1000 dilution) or b-actin (1:2000 dilution) at 4 C overnight. Afterwards, the membrane was washed and
DOI: 10.3109/08923973.2015.1009997
Osthole inhibits inflammatory cytokine release through PPAR / -mediated mechanisms
incubated with fluorescent secondary antibody for 1 h at room temperature. The ratio of the protein interested was subjected to b-actin and was densitometrically analyzed by the Odyssey infrared imaging system. Statistical analysis
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Data are expressed as mean ± SD of at least three independent experiments. The one-way ANOVA followed by a post hoc LSD test was used for comparisons between groups. The statistical analysis was conducted using the SPSS 18.0 program (SPSS Inc., Chicago, IL), and differences were considered at a value of p50.0 5.
Results Effect of osthole on cell viability The goal of this study was to investigate the effect of osthole on the inflammatory cytokine release in cultured 3T3-L1 adipocytes, the dosage of osthole should not affect cell viability. Therefore, we examined the cytotoxicity of osthole by MTT assay. The results revealed that the cell survival rate did not differ obviously when 3T3-L1 adipocytes were treated with osthole 0.1, 0.4, 1.6, and 6.4 mM for 3, 6, 12, 24, and 48 h, respectively (Figure 1), indicating that osthole, in concentrations from 0.1 to 6.4 mM, had no cytotoxicity on 3T3-L1 adipocytes, and the concentrations of osthole might be used in the experiments. Effects of osthole on TNF-a and IL-6 levels in cultured supernatants of 3T3-L1 adipocytes To determine whether osthole might affect the inflammatory cytokine production in normal adipocytes, the TNF-a and
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IL-6 levels in cultured supernatants were measured by the ELISA method. The experimental results showed that the TNF-a and IL-6 levels between control and vehicle groups were not significantly different (Figure 2), indicating that 1% DMSO did not affect the synthesis and release of these cytokines in cultured adipocytes. Following treatment of adipocytes with osthole 0.1–1.6 mM for 8 h, the TNF-a and IL-6 levels in cultured supernatants also exhibited no obvious changes as compared with the vehicle group (Figure 2), suggesting that osthole treatment might not alter the background levels of TNF-a and IL-6 in normal adipocytes under current treatment conditions. Effects of osthole on TNF-a and IL-6 levels in cultured supernatants of 3T3-L1 adipocytes stimulated with LPS LPS may potently induce adipocyte activation and the production of inflammatory cytokines. To examine the effects of osthole on the synthesis and release of TNF-a and IL-6 in activated adipocytes, LPS was used in the experiments. The present results showed that after stimulation with LPS 2 mg/ml for 6 h, the levels of TNF-a and IL-6 in cultured supernatants were significantly increased as compared with the vehicle group (p50.05, Figure 3). The addition of osthole could prevent the increments of TNF-a and IL-6 levels. As shown in Figure 3, the TNF-a level was suppressed by 6.5% at 0.1 mM, 20.8% at 0.4 mM, and 56.0% at 1.6 mM, and the effect showed a good dose–effect relationship. Also, the IL-6 level was inhibited by 44.4% at 0.1 mM, 38.3% at 0.4 mM, and 28.0% at 1.6 mM osthole, but the dose–effect relationship was not observed, the most effective concentration of osthole was 0.1 mM (p50.01, Figure 3).
Figure 1. Cell viability after treatment with osthole 0.1–6.4 mM for 3–48 h. Data are presented as mean ± SD, with n ¼ 4 per group.
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Figure 2. The TNF-a and IL-6 levels in cultured supernatants after treatment of 3T3-L1 adipocytes with osthole for 8 h. Data are presented as mean ± SD, with n ¼ 4 per group.
Figure 3. The TNF-a and IL-6 levels in cultured supernatants of 3T3-L1 adipocytes stimulated with LPS 2 mg/ml for 6 h after pretreatment with osthole for 2 h. Data are presented as mean ± SD, with n ¼ 4 per group. *p50.05 versus the vehicle group; #p50.05, ##p50.01 versus the LPS 2 mg/ml group.
Effects of osthole on PPARa/c and NF-kB p65 protein expressions in 3T3-L1 adipocytes stimulated with LPS To investigate whether the anti-inflammatory effect of osthole was associated with PPARa/g-NF-kB pathways, the protein levels of PPARa/g and NF-kB expressions in adipocytes were determined by Western blot assay. The results are shown in Figure 4. Compared with the vehicle group, the protein expressions of PPARa and NF-kB p65 in 3T3-L1 adipocytes in the LPS-stimulated group was low and high (p50.01), but the protein expression of PPARg was unchanged. The addition of 0.1–1.6 mM osthole could dose dependently increase the PPARa/g protein expressions
(Figure 4a and b) and decrease the NF-kB p65 protein expression (Figure 4c) in 3T3-L1 cells stimulated with LPS (p50.05 or p50.01). Effects of PPAR a/c inhibitors on osthole-reduced TNF-a and IL-6 levels in cultured supernatants of 3T3-L1 adipocytes stimulated with LPS In order to further confirm whether osthole reduced TNF-a and IL-6 production via PPARa/g pathways, a PPARa inhibitor MK886 (4 mM) and a PPARg inhibitor GW9662 (10 mM) were applied. After pretreatment of adipocytes with MK886 for 2 h, the inhibitory effects of osthole on TNF-a and
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DOI: 10.3109/08923973.2015.1009997
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Figure 4. The PPARa/g and NF-kB p65 protein expressions in 3T3-L1 adipocytes stimulated with LPS 2 mg/ml for 6 h after pretreatment with osthole for 2 h. Data are presented as mean ± SD, with n ¼ 3 per group. **p50.01 versus the vehicle group; #p50.05, ##p50.01 versus the LPS 2 mg/ml group.
IL-6 were attenuated (p50.05, Figure 5a), but not completely halted (p50.05, Figure 5a). Likewise, after pretreatment of adipocytes with GW9662 or MK886 plus GW9662 for 2 h, the inhibitory effects of osthole on TNF-a and IL-6 were notably alleviated or almost cancelled (p50.05, Figure 5b and c). The effects of MK886 and/or GW9662 per se on the two inflammatory cytokines were not found. Effects of PPAR a/c inhibitors on osthole-reduced NF-iB p65 protein expression in 3T3-L1 adipocytes stimulated with LPS NF-kB is known to be required for the induction of inflammatory cytokines, PPAR a/g may affect its expression. The present experimental results showed that after pretreatment of 3T3-L1 adipocytes with PPAR a inhibitor MK886 for 2 h, the osthole-reduced protein expression of NF-kB p65 was attenuated (p50.05, Figure 6a), but not completely halted (p50.05, Figure 6a). After the cells were pretreated with PPAR g inhibitor GW9662 or MK886 plus GW9662 for 2 h, the inhibitory effect of osthole on NF-kB p65 protein expression was almost cancelled (p50.05, Figure 6b and c). The effects of MK886 and/or GW9662 per se on NF-kB p65 protein expression were not found.
Discussion Recent literature data show that circulating inflammatory cytokines are elevated in patients with NASH24,25, and the origination from adipose tissue may contribute about 30%26. These inflammatory cytokines not only induce the ‘‘second hits’’ of liver but also enhance the hepatic lipid accumulation via induction of insulin resistance27–29. Therefore, the reduction of inflammatory cytokines in adipose tissue is very important to amelioration of NASH. Our present results showed that osthole could decrease the TNF-a and IL-6 levels in cultured supernatants of 3T3-L1 adipocytes stimulated with LPS, suggesting that the therapeutic effect of osthole on
NASH might be partly attributable to its inhibitory effect on TNF-a and IL-6 synthesis and release in adipose tissue. However, we found that the inhibitory effect of osthole on IL-6 was decreased with increasing concentrations of osthole. The potential reason might be the enhancement of ERK1/2 phosphorylation by osthole30, which might result in the increment of IL-6 release. Several studies in the experimental models indicate that activation of PPARa/g can inhibit the generation of inflammatory cytokines through interference with the inflammatory transcription factor NF-kB14,31. In order to explore the possible mechanisms of osthole in inhibiting TNF-a and IL-6 production, we examined the effects on PPARa/g and NF-kB p65 protein expressions in cultured adipocytes. The present results, in accordance with the results of mRNA expressions23, showed that osthole could increase the PPARa/g protein expressions in a dose-dependent manner. Accordingly, the NF-kB p65 protein expression was decreased, and the finding was also consistent with the result of in vitro cultured macrophages30. So, we thought that inhibitory effects of osthole on TNF-a and IL-6 might be related to reduction of NF-kB inflammatory signaling pathway via activation of PPARa/g. But it is necessary to investigate the exact effect of osthole on the modulation of NF-kB. If so, how osthole affects the nuclear translocation or its activity is a valuable issue to research further. In order to further verify whether osthole suppressed the TNF-a and IL-6 release from LPS-stimulated adipocytes through PPARa/g-NF-kB pathways, a specific PPARa inhibitor MK886 and a specific PPARg inhibitor GW9662 were used32,33. The results showed that after pretreatment of adipocytes with MK886 and/or GW9662 for 2 h, the inhibitory effects of osthole on TNF-a and IL-6 were decreased or almost cancelled, and the effects on NF-kB p65 protein expression also exhibited similar variations. These results revealed that PPARa/g-NF-kB signaling pathways involved in the inhibitory effects of osthole on TNF-a and IL-6 production in adipocytes. But our experimental results disclosed that
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Figure 5. Effects of osthole on TNF-a and IL-6 levels in cultured supernatants of 3T3-L1 adipocytes stimulated with LPS after the cells were pretreated with PPARa inhibitor MK886 4 mM (a), PPARg inhibitor GW9662 10 mM (b), and PPARa inhibitor MK886 4 mM plus PPARg inhibitor GW9662 10 mM (c) for 2 h. Data are presented as mean ± SD, with n ¼ 3 per group. *p50.05, **p50.01 versus the vehicle group; #p50.05, ##p50.01 versus the LPS 2 mg/ml group; &p50.05, &&p50.01 versus the GW9662 and/or MK886 group; 4p50.05 versus the osthole 1.6 mM + LPS 2 mg/ml group; mp50.05 versus the MK886 4 mM + LPS 2 mg/ml group.
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DOI: 10.3109/08923973.2015.1009997
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Figure 6. Effect of osthole on NF-kB p65 protein expression in 3T3-L1 adipocytes stimulated with LPS after the cells were pretreated with PPARa inhibitor MK886 4 mM (a), PPARg inhibitor GW9662 10 mM (b), and PPARa inhibitor MK886 4 mM plus PPARg inhibitor GW9662 10 mM (c) for 2 h. Data are presented as mean ± SD, with n ¼ 3 per group. **p50.01 versus the vehicle group; #p50.05 versus the LPS 2 mg/ml group; &&p50.01 versus the GW9662 and/or MK886 group; 4p50.05 versus the osthole 1.6 mM + LPS 2 mg/ml group; mp50.05 versus the MK886 4 mM + LPS 2 mg/ml group.
the pretreatment with 4 mM MK886 did not completely cancelled the inhibitory effects of osthole on the inflammatory cytokine release and NF-kB protein expression, suggesting that the concentration of MK886 used in this study might not be high enough and the osthole still worked under the conditions. In summary, our results further demonstrated that osthole could inhibit the TNF-a and IL-6 production in adipocytes, and the inhibitory effects might be related to reduction of NF-kB expression via activation of PPARa/g. This may be a novel pharmacological effect of osthole in the reduction of inflammatory response and insulin resistance, and be good for the prevention and treatment of NASH.
Declaration of interest This research was sponsored by the National Natural Science Foundation of China (No. 81173067), the Postgraduate Innovative Foundation of Jiangsu Province (No. CXLZ120854), the Excellent Postgraduate Items of Soochow University, and the Priority Academic Program Development of Jiangsu Higher Education Institutions, China. The authors declare no conflicts of interest.
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