International Immunopharmacology 21 (2014) 354–360
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Emodin inhibits LPS-induced inflammatory response by activating PPAR-γ in mouse mammary epithelial cells Zhengtao Yang 1, Ershun Zhou 1, Dong Wei, Depeng Li, Zhengkai Wei, Wen Zhang, Xichen Zhang ⁎ College of Veterinary Medicine, Jilin University, Changchun 130062, Jilin Province, PR China
a r t i c l e
i n f o
Article history: Received 8 February 2014 Received in revised form 12 May 2014 Accepted 13 May 2014 Available online 27 May 2014 Keywords: Emodin NF-κB PPAR-γ Cytokines LPS Mastitis
a b s t r a c t Emodin, an anthraquinone derivative isolated from the rhizomes of Rheum palmatum, has been reported to have a protective effect against lipopolysaccharide (LPS)-induced mastitis. However, the underlying molecular mechanisms are not well understood. The aim of this study was to investigate the molecular mechanisms of emodin in modifying lipopolysaccharide (LPS)-induced signaling pathways in mouse mammary epithelial cells (MEC). The pro-inflammatory cytokines were determined by ELISA. Nuclear factor-κB (NF-κB), inhibitory kappa B (IκBα) protein, p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and PPAR-γ were determined by Western blotting. The results showed that emodin suppressed tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), iNOS and COX-2 expression. We also found that emodin inhibited LPS-induced NF-κB activation, IκBα degradation, phosphorylation of ERK, JNK and P38. Furthermore, emodin could activate PPAR-γ and the anti-inflammatory effects of emodin can be reversed by GW9662, a specific antagonist for PPAR-γ. In conclusion, our results demonstrate that emodin activates PPAR-γ, thereby attenuating LPS-induced inflammatory response. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Inflammation, characterized by redness, swelling, heat, pain and dysfunction of the organs, is a fundamental protective response that sometimes goes awry and becomes a major cofactor in the pathogenesis of many diseases, such as mastitis [1,2]. Inflammation is usually caused by various factors such as microbial infection, tissue damage, and cardiac infarction. LPS, a potent virulence component from Gram negative bacteria, is a well-known inducer of the innate immune response and has been reported to be an important trigger of inflammation [3,4]. The innate immune system is the major contributor to acute inflammation induced by microbial infection or tissue damage [5,6]. Besides macrophages and dendritic cells (DCs), nonprofessional cells also play important roles such as epithelial cells, endothelial cells, and fibroblasts to innate immunity. Mammary epithelial cells (MEC) are the first line of the mammary gland in defensing the invading pathogens [7]. In vitro culture of MEC is widely used as a model to study the capacity of cells to sense and respond to mastitis-causing bacteria. Emodin (3-methyl-1, 6, 8-trihydroxyanthraquinone) is an anthraquinone derivative from the rhizome of Rheum palmatum that is widely used as a laxative in traditional Chinese medicine [8]. It has been reported that emodin shows a number of biological activities
⁎ Corresponding author at: Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun 130062, PR China. Tel./fax: + 86 431 87981351. E-mail address: [email protected] (X. Zhang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2014.05.019 1567-5769/© 2014 Elsevier B.V. All rights reserved.
such as immunosuppressive, hepatoprotective, anti-tumor and antiinflammatory activities [9–13]. Treatment of RAW 264.7 macrophages with emodin suppressed the expression of a panel of inflammatoryassociated genes, including tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), as well as the nuclear translocation of nuclear factor-κB (NF-κB) [14]. Recently, it has been reported to have a protective effect against lipopolysaccharide (LPS)-induced mastitis. However, the underlying molecular mechanisms are not well understood. Mammary epithelial cells have been reported to play an important role in defensing the invading pathogens. Thus, in the present study, LPS-stimulated mouse MEC was used as a tool to investigate the anti-inflammatory effect of emodin and elucidate the potential mechanisms. 2. Materials and methods 2.1. Materials Emodin (3-methyl-1, 6, 8-trihydroxyanthraquinone) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Jilin, China) (Fig. 1). LPS (Escherichia coli 055:B5) was purchased from Sigma (St. Louis, MO, USA). Dulbecco's modified eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen-Gibco (Grand Island, NY). Mouse TNF-α, IL-6 and IL-1β enzyme-linked immunosorbent assay (ELISA) kits were purchased from Biolegend (CA, USA). Rabbit monoclonal antibodies IκBα, p65, p-p65, ERK, p-ERK, p38, p-p38, JNK, p-JNK, iNOS, COX-2 and mouse monoclonal antibodies p-IκBα were purchased from Cell Signaling
Z. Yang et al. / International Immunopharmacology 21 (2014) 354–360
355
40 μg/ml for 18 h. Cell-free supernatants were collected for the proinflammatory cytokines assays with a mouse ELISA kit, according to the manufacturer's instructions (Biolegend, Inc, San Diego, CA, USA). 2.6. Western blot analysis
Fig. 1. Chemical structure of emodin.
Technology Inc (Beverly, MA, USA). Mouse mAb PPAR-γ was purchased from ABCAM. Horseradish peroxidase-conjugated goat anti-rabbit and goat-mouse antibodies were provided by GE Healthcare (Buckinghamshire, UK). All other chemicals were of reagent grade. 2.2. Animals BALB/c mice (8–12 day gravid), weighting approximately 20–24 g, were purchased from the Center of Experiment Animals of Baiqiuen Medical College of Jilin University (Jilin, China). All animal experiments were performed in accordance with the National Institutes of Health guide for the Care and Use of Laboratory Animal. All female and male mice were housed together respectively for 2–3 d to adapt themselves to the surroundings. Then 2 female and 1 male mice were randomly divided in each cage supplied with sufficient water and forages. All cages had been washed carefully and conducted with autoclave sterilization. The house had been sterilized thoroughly with disinfectants.
MEC (1 × 106) were seeded in 6-well plates. When the plate was confluent, MEC were pretreated with emodin for 1 h before LPS stimulation. After LPS stimulation for 1 h, the cells were collected and washed twice with ice-cold PBS. Total proteins from cells were extracted by M-PER Mammalian Protein Extraction Reagent (Thermo). Protein concentration was determined by the BCA method. Equal amounts of protein were loaded in each well and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which subsequently was transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked for 2 h with 5% skim milk in TBST on the shaker at room temperature and then washed 3 times for each time 10 min with Tri–Tween buffered saline (TTBS, 20 mM Tris–HCl buffer, pH 7.6, 137 mM NaCl and 0.05% Tween 20). The membrane was placed on Primary antibody diluted at a 1:1000 proportion in diluent buffer [5% (w/v) BSA and 0.1% Tween 20 in TBS] and incubated overnight at 4 °C on the shaker. Subsequently, the membrane was washed with PBS-T followed by incubation with the secondary antibody conjugated with horse-radish peroxidase at room temperature for 1 h. The membrane was again washed 3 times for 10 min each time as above and finally the results were generated by using an enhanced chemiluminescence (ECL) Western blotting kit. 2.7. PPAR-γ ligand binding assay
2.3. Culture of mouse MEC Primary cultured mMECs were prepared as previously described by Smalley. Briefly, the mammary glands were collected from BALB/c mice under sterile conditions in the middle of pregnancy. Then the mammary tissues were minced into pasty fine tissues and digested by collagenase I/II/trypsin mixture and shaken at 37 °C. After filtration to remove undissociated tissue and debris, the cells were collected by centrifugation at 250 g for 5 min three times. The cell pellets were resuspended in DMEM/F12 containing 10% FCS and incubated for 1 h at 37 °C, and then the supermatant was collected. This step was repeated three times until the fibroblasts were removed. After the last incubate, the cells were resuspended in DMEM/F12 containing 10% FCS, 0.5% transferrin, 0.1% T3, and 0.5% EGF, and were cultured at 37 °C. The culture media were changed once every 24 h. The mMECs were identified by cytokeratin-18 immunocytochemistry as described by our previously studies [15]. When the plate was confluent, MEC were incubated in the presence or absence of different concentrations of emodin at 1 h before LPS challenge.
The ligand binding activity was detected using a NuLigand PPAR-γ kit (Microsystems, Kyoto, Japan) according to the manufacturer's instruction. The NuLigand PPAR-γ kit is a cell free assay based on the ligand-dependent interaction between PPAR-γ and TIF-2, so called CoA–BAP system [16]. 2.8. PPAR-γ activation and inhibition To test the activation of PPAR-γ by emodin, the MEC were pretreated with emodin for 1 h before LPS incubation. After LPS (1 μg/ml)
2.4. Cell viability assay Cell viability was measured by MTT assay. The cells were plated at a density of 4 × 105 cells/ml in 96-well plates at 37 °C with 5% CO2 for 1 h and then treated with 50 μl emodin at different concentrations (0–40 μg/ml) for 1 h followed by stimulation with 50 μl LPS. 18 h of LPS stimulation later, 20 μl MTT (5 mg/ml) was added to each well and the cells were again incubated for 4 h. The supernatant was removed and the formation of formazan was resolved with 150 μl of DMSO each well. The optical density was measured at 570 nm on a microplate reader (TECAN, Austria). 2.5. ELISA assay 5
MEC were seeded in 24-well plates (10 cell/well) and incubated in the presence of either LPS 1 μg/ml alone or LPS plus emodin 10, 20, and
Fig. 2. Effect of emodin on the cell viability of mouse mammary epithelial cells. Cells were cultured with different concentrations of emodin (0–40 μg/ml) in the absence or presence of 1 mg/L LPS for 24 h. The cell viability was determined by MTT assay. The values presented are the means ± SEM of three independent experiments.
356
Z. Yang et al. / International Immunopharmacology 21 (2014) 354–360
Fig. 3. Emodin inhibits lipopolysaccharide (LPS)-induced cytokine production in a dose-dependent manner. Cells were treated with 1 μg/ml LPS in the absence or presence of emodin (10, 20, 40 μg/ml) for 24 h. Levels of TNF-α (A) and IL-6 (B) in culture supernatants were measured by ELISA. The data presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #p b 0.05 vs. control group; *p b 0.05, **p b 0.01 vs. LPS group.
stimulation for 9 h, the expression of PPAR-γ was detected by Western blotting. Meanwhile, to determine whether the anti-inflammatory effect of emodin is dependent on PPAR-γ activation, GW9662, the specific
PPAR-γ antagonist was added into the medium 30 min before emodin incubation. Then the cells were stimulated by LPS and NF-κB activation and the expression of inflammatory cytokines was detected.
Fig. 4. Emodin inhibits lipopolysaccharide (LPS)-induced NF-κB activation and IκBα degradation. Cells were preincubated with emodin (10, 20, 40 μg/ml) for 1 h and then treated with 1 μg/ml LPS for 1 h. Protein samples were analyzed by Western blot with specific antibodies. β-actin was used as a control. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #p b 0.05 vs. control group; *p b 0.05, **p b 0.01 vs. LPS group.
Z. Yang et al. / International Immunopharmacology 21 (2014) 354–360
2.9. Statistical analysis All data are expressed as mean ± S.E.M. The differences among the various experimental groups were analyzed by one-way ANOVA. P b 0.05 was considered to be statistically significant. 3. Results
357
anti-inflammatory effects of emodin in LPS-induced mouse MEC, the expressions of iNOS, COX-2, TNF-α and IL-6 were measured. The results showed that emodin alone did not induce the expressions of iNOS, COX-2, TNF-α and IL-6 compared with those in the control group. LPS caused a significant increase of the expressions of iNOS, COX-2, TNF-α and IL-6 compared with those in the control group (P b 0.01). Emodin markedly reduced the concentration of these cytokines at a dosedependent manner (Fig. 3).
3.1. Effects of emodin on cell viability To assess whether emodin exerted the potential cytotoxicity effect on mouse MEC, the cells were evaluated by the MTT assay after incubated for 18 h in the absence or presence of LPS. Our result displayed that emodin had no significant effects on cell viability at a dose-dependent concentrations (10, 20, 40 μg/ml) (Fig. 2). In other words, the viability of the cells was not nearly affected by the treatment with emodin. 3.2. Emodin decreased LPS-induced up-regulation of inflammatory mediators in LPS-induced mouse MEC TNF-α, IL-6, iNOS and COX-2 are major inflammatory mediators and play an important role in inflammatory diseases [17,18]. To evaluate the
3.3. Emodin blocked LPS-induced NF-κB pathway NF-κB is a multi-subunit nuclear transcription factor which rapidly activates the transcription of various cytokines and chemokines and plays a critical role in the inflammatory response [19,20]. In resting cells, NF-κB is bound to its inhibitor IκB in the cytoplasm. Once stimulated by LPS, IκB is phosphorylated and degraded, and then the released NF-κB is transferred into the nucleolus and up-regulates the transcription of the inflammatory factors [21]. To explore whether the antiinflammatory effects of emodin are mediated by NF-κB pathway in LPS-induced mouse MEC, the levels of NF-κB and IκBα were determined through Western blot. The results indicated that emodin significantly suppressed the activation of NF-κB and the degradation of IκBα (Fig. 4).
Fig. 5. Emodin inhibits lipopolysaccharide (LPS)-induced MAPK activation. Cells were preincubated with emodin (10, 20, 40 μg/ml) for 1 h and then treated with 1 μg/ml LPS for 1 h. Protein samples were analyzed by Western blot with specific antibodies. β-actin was used as a control. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #p b 0.05 vs. control group; *p b 0.05, **p b 0.01 vs. LPS group.
358
Z. Yang et al. / International Immunopharmacology 21 (2014) 354–360
3.4. Emodin inhibited LPS-induced MAPK pathway The mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in regulating gene expression in eukaryotic cells and links extracellular signals to the machinery that controls fundamental cellular processes such as growth, proliferation, differentiation, migration, and apoptosis [22,23]. To certify whether the MAPK pathway is involved in the anti-inflammatory effects of emodin, the levels of p38, JNK, and ERK MAPKs were detected through Western blot. The results demonstrated that emodin markedly inhibited the LPS-induced increase of phosphorylation of p38, ERK, and JNK in a dose-dependent manner (Fig. 5). 3.5. Emodin was a PPAR-γ ligand To test whether emodin served as a PPAR-γ ligand, we detected PPAR-γ ligand binding activity using the CoA–BAP system. As shown in Fig. 6, emodin showed a dose-dependent PPAR-γ ligand binding activity. It was suggested that emodin was a PPAR-γ ligand. 3.6. Effects of emodin on PPAR-γ activation Some studies have shown that some herbal products exert antiinflammatory effects by activating PPAR-γ [24–26]. In this study, we investigated the effects of emodin on PPAR-γ activation. The results showed that emodin up-regulated the expression of PPAR-γ in a dose manner (Fig. 7). 3.7. Effects of emodin on LPS-induce inflammatory response is PPAR-γ dependent As shown in Fig. 8, LPS increased the expression of TNF-α and IL-6 and emodin reduced TNF-α and IL-6 expression dramatically. Emodin inhibited NF-κB activation induced by LPS. Whereas, GW9662, the PPAR-γ specific antagonist could abolish the anti-inflammatory effect of emodin. 4. Discussion In the present study, the anti-inflammatory effects of emodin on LPS-stimulated mouse MEC were assessed. Our results showed that emodin clearly inhibited the expression of pro-inflammatory mediators
Fig. 6. PPAR-γ ligand binding activity of emodin. The binding affinity of emodin towards the PPAR-γ LBD at the indicated concentration was measured using a NuLigand PPAR-γ kit. The control group was expressed as ‘1’ and fold change was calculated as other groups vs control group. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. *p b 0.05, **p b 0.01 vs. control group.
Fig. 7. Effects of emodin on PPAR-γ expression in mouse mammary epithelial cells. Cells were pretreated with different concentrations of emodin (10, 20, 40 μg/ml) for 9 h. Protein samples were analyzed by western blot with specific antibodies. β-actin was used as a control. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. *p b 0.05, **p b 0.01 vs. control group.
and suppressed NF-κB and MAPK activation in LPS-stimulated mouse MEC. Furthermore, emodin could activate PPAR-γ and the antiinflammatory effects of emodin can be reversed by GW9662, a specific antagonist for PPAR-γ. It is suggested that emodin activates PPAR-γ, thereby attenuating LPS-induced inflammatory response. Inflammation is a protective response by the body to ensure removal of harmful stimuli as well as a healing process for repairing damaged tissue [27]. However, overproduction of pro-inflammatory cytokines by immune cells to overwhelm pathogens can be fatal. During infection, the inflammatory responses are regulated by the cytokine network [28]. It is well known that TNF-α and IL-6 involved in the initiation and development of acute inflammation are important inflammatory mediators released in response to various injurious stimuli. LPS, a potent stimuli, can arouse the production of TNF-α and IL-6 through activating host immune and inflammation cells. TNF-α is called primary cytokine, for it exerts an important role in initiating an acute inflammatory response [29]. TNF-α can induce infiltration and activation of neutrophils, impair vascular endothelial cells, up-regulate cellular adhesion molecules, promote the secretion of oxygen-derived free radicals and initiate and aggravate the cascade of other inflammatory mediators. IL-6 is a pleiotropic cytokine produced by the cells of immune and nonimmune origin. Increased production of IL-6 is associated with disturbances of homeostasis, such as trauma, sepsis, or inflammatory diseases [30]. Previous studies demonstrated that emodin could inhibit TNF-α and IL-6 in LPS-stimulated human umbilical vein endothelial cells [31]. In this study, our results showed that emodin markedly decreased the levels of TNF-α and IL-6 in a dose-dependent manner in LPS-stimulated mouse MEC. It suggested that emodin attenuated the inflammatory responses through inhibiting the production of pro-inflammatory cytokines. Studies have shown that the production of TNF-α and IL-6 is regulated by NF-κB and MAPKs. To further explore the anti-inflammatory mechanism of emodin, NF-κB and MAPK activation was detected. NF-κB is a ubiquitously expressed family of transcription factors controlling the expression of numerous genes involved in inflammatory and immune responses and cellular proliferation [32]. In resting
Z. Yang et al. / International Immunopharmacology 21 (2014) 354–360
359
Fig. 8. Effects of emodin on LPS-induced inflammatory response is PPAR-γ dependent. Cells were treated with 40 μg/ml emodin for 1 h, or 10 μM GW9662 for 30 min before emodin incubation, and stimulated with LPS for 24 h. Protein samples were analyzed by Western blot with NF-κB antibody. Levels of TNF-α (A) and IL-6 (B) in culture supernatants were measured by ELISA. The data presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #p b 0.05 vs. control group; *p b 0.05, **p b 0.01 vs. LPS group.
cells, NF-κB is sequestered in the cytoplasm by association with inhibitory proteins called IκBs. Once stimulated by LPS, IκB is then ubiquitinated and degraded by the proteasome, freeing NF-κB to be translocated into the nucleus and to exert its functions as transcription factor. Our data displayed that emodin obviously suppressed NF-κB activation and IκB degradation in LPS-stimulated mouse MEC. We also found that emodin significantly inhibited the phosphorylation of ERK, JNK, and p38 in LPS-induced mouse MEC. Taken together, our data revealed that emodin blocked the activation of NF-κB and MAPK signal pathways. Therefore, emodin may decrease the inflammatory response through blocking the activation of NF-κB and MAPK signal pathways. Peroxisome proliferator-activated receptors (PPARs) are ligandactivated transcription factors that play important roles in the regulation of a large number of biological processes including inflammation. Ligands for PPAR-γ have been reported to exert negative effects on inflammation and immune responses. Some studies have shown that some herbal products exert anti-inflammatory effects by activating PPAR-γ. Reports have shown that PPAR-γ agonists could negatively regulate LPS-induced inflammatory responses [33]. Thus, the effects of emodin on PPAR-γ expression were detected. In this study, we found that emodin could up-regulate the expression of PPAR-γ in a dose manner. We also found that the inhibitory effect of TNF-α and IL-6 by emodin can be reversed by GW9662, the PPAR-γ specific antagonist. Our results suggested that the ability of attenuating inflammation by emodin in mouse MEC was dependent on the PPAR-γ pathway. In summary, this study has demonstrated that emodin decreased the levels of the pro-inflammatory cytokines and suppressed NF-κB and MAPK signal pathways in LPS-induced mouse MEC. The promising anti-inflammatory mechanism of emodin may be that emodin activates PPAR-γ, thereby attenuating LPS-induced NF-κB and MAPK activation and the release of pro-inflammatory cytokines.
Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (Nos. 30972225 and 30771596).
References [1] Conese M, Assael BM. Bacterial infections and inflammation in the lungs of cystic fibrosis patients. Pediatr Infect Dis J 2001;20:207–13. [2] Ben-Neriah Y, Karin M. Inflammation meets cancer, with NF-[kappa] B as the matchmaker. Nat Immunol 2011;12:715–23. [3] Atabai K, Matthay MA. The pulmonary physician in critical care. 5: Acute lung injury and the acute respiratory distress syndrome: definitions and epidemiology. Thorax 2002;57:452–8. [4] Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, et al. Incidence and outcomes of acute lung injury. N Engl J Med 2005;353:1685–93. [5] Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006;124:783–801. [6] Beutler B, Jiang Z, Georgel P, Crozat K, Croker B, Rutschmann S, et al. Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large. Annu Rev Immunol 2006;24:353–89. [7] Strandberg Y, Gray C, Vuocolo T, Donaldson L, Broadway M, Tellam R. Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells. Cytokine 2005;31:72–86. [8] Shi YQ, Fukai T, Sakagami H, Kuroda J, Miyaoka R, Tamura M, et al. Cytotoxic and DNA damage-inducing activities of low molecular weight phenols from rhubarb. Anticancer Res 2001;21:2847–53. [9] Huang HC, Lee CR, Chao PD, Chen CC, Chu SH. Vasorelaxant effect of emodin, an anthraquinone from a Chinese herb. Eur J Pharmacol 1991;205:289–94. [10] Kuo YC, Meng HC, Tsai WJ. Regulation of cell proliferation, inflammatory cytokine production and calcium mobilization in primary human T lymphocytes by emodin from Polygonum hypoleucum Ohwi. Inflamm Res 2001;50:73–82. [11] Lin CC, Chang CH, Yang JJ, Namba T, Hattori M. Hepatoprotective effects of emodin from Ventilago leiocarpa. J Ethnopharmacol 1996;52:107–11. [12] Chang CJ, Ashendel CL, Geahlen RL, McLaughlin JL, Waters DJ. Oncogene signal transduction inhibitors from medicinal plants. In Vivo 1996;10:185–90. [13] Huang Z, Chen G, Shi P. Effects of emodin on the gene expression profiling of human breast carcinoma cells. Cancer Detect Prev 2009;32:286–91. [14] Li HL, Chen HL, Li H, Zhang KL, Chen XY, Wang XW, et al. Regulatory effects of emodin on NF-kappaB activation and inflammatory cytokine expression in RAW 264.7 macrophages. Int J Mol Med 2005;16:41–7. [15] Fu Y, Zhou E, Wei Z, Liang D, Wang W, Wang T, et al. Glycyrrhizin inhibits the inflammatory response in mouse mammary epithelial cells and mouse mastitis model. FEBS J 2014. [16] Kanayama T, Mamiya S, Nishihara T, Nishikawa J. Basis of a high-throughput method for nuclear receptor ligands. J Biochem 2003;133:791–7. [17] Satoh H, Firestein GS, Billings PB, Harris JP, Keithley EM. Proinflammatory cytokine expression in the endolymphatic sac during inner ear inflammation. J Assoc Res Otolaryngol 2003;4:139–47. [18] Libby P. Coronary artery injury and the biology of atherosclerosis: inflammation, thrombosis, and stabilization. Am J Cardiol 2000;86:3J–8J (discussion J-9J). [19] Caamano J, Hunter CA. NF-kappaB family of transcription factors: central regulators of innate and adaptive immune functions. Clin Microbiol Rev 2002;15:414–29.
360
Z. Yang et al. / International Immunopharmacology 21 (2014) 354–360
[20] in Csl-iimbinf-kBa, in RmCsl-iimbinf-kBa, RAW 264.7 macrophagesVallabhapurapu S, Karin M. Regulation and function of NF-kappaB transcription factors in the immune system. Annu Rev Immunol 2009;27:693–733. [21] Choi YH, Jin GY, Li GZ, Yan GH. Cornuside suppresses lipopolysaccharide-induced inflammatory mediators by inhibiting nuclear factor-kappa B activation in RAW 264.7 macrophages. Biol Pharm Bull 2011;34:959–66. [22] Mordret G. MAP kinase kinase: a node connecting multiple pathways. Biol Cell 1993;79:193–207. [23] Jebamercy G, Vigneshwari L, Balamurugan K. A MAP Kinase pathway in Caenorhabditis elegans is required for defense against infection by opportunistic Proteus species. Microbes Infect 2013;15:550–68. [24] Lim HA, Lee EK, Kim JM, Park MH, Kim DH, Choi YJ, et al. PPARgamma activation by baicalin suppresses NF-kappaB-mediated inflammation in aged rat kidney. Biogerontology 2012;13:133–45. [25] Park MY, Lee KS, Sung MK. Effects of dietary mulberry, Korean red ginseng, and banaba on glucose homeostasis in relation to PPAR-alpha, PPAR-gamma, and LPL mRNA expressions. Life Sci 2005;77:3344–54. [26] Song MK, Roufogalis BD, Huang TH. Modulation of diabetic retinopathy pathophysiology by natural medicines through PPAR-gamma-related pharmacology. Br J Pharmacol 2012;165:4–19.
[27] Medzhitov R. Origin and physiological roles of inflammation. Nature 2008;454:428–35. [28] Lee JW, Bannerman DD, Paape MJ, Huang MK, Zhao X. Characterization of cytokine expression in milk somatic cells during intramammary infections with Escherichia coli or Staphylococcus aureus by real-time PCR. Vet Res 2006;37:219–29. [29] Skotnicka B, Hassmann E. Proinflammatory and immunoregulatory cytokines in the middle ear effusions. Int J Pediatr Otorhinolaryngol 2008;72:13–7. [30] Kishimoto T, Akira S, Taga T. Interleukin-6 and its receptor: a paradigm for cytokines. Science 1992;258:593–7. [31] Meng G, Liu Y, Lou C, Yang H. Emodin suppresses lipopolysaccharide-induced proinflammatory responses and NF-kappaB activation by disrupting lipid rafts in CD14-negative endothelial cells. Br J Pharmacol 2010;161:1628–44. [32] Ghosh S, Karin M. Missing pieces in the NF-kappaB puzzle. Cell 2002;109(Suppl.): S81–96. [33] Li H, Ruan XZ, Powis SH, Fernando R, Mon WY, Wheeler DC, et al. EPA and DHA reduce LPS-induced inflammation responses in HK-2 cells: evidence for a PPAR-gamma-dependent mechanism. Kidney Int 2005;67:867–74.
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Emodin inhibits LPS-induced inflammatory response by activating PPAR-γ in mouse mammary epithelial cells Zhengtao Yang 1, Ershun Zhou 1, Dong Wei, Depeng Li, Zhengkai Wei, Wen Zhang, Xichen Zhang ⁎ College of Veterinary Medicine, Jilin University, Changchun 130062, Jilin Province, PR China
a r t i c l e
i n f o
Article history: Received 8 February 2014 Received in revised form 12 May 2014 Accepted 13 May 2014 Available online 27 May 2014 Keywords: Emodin NF-κB PPAR-γ Cytokines LPS Mastitis
a b s t r a c t Emodin, an anthraquinone derivative isolated from the rhizomes of Rheum palmatum, has been reported to have a protective effect against lipopolysaccharide (LPS)-induced mastitis. However, the underlying molecular mechanisms are not well understood. The aim of this study was to investigate the molecular mechanisms of emodin in modifying lipopolysaccharide (LPS)-induced signaling pathways in mouse mammary epithelial cells (MEC). The pro-inflammatory cytokines were determined by ELISA. Nuclear factor-κB (NF-κB), inhibitory kappa B (IκBα) protein, p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and PPAR-γ were determined by Western blotting. The results showed that emodin suppressed tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), iNOS and COX-2 expression. We also found that emodin inhibited LPS-induced NF-κB activation, IκBα degradation, phosphorylation of ERK, JNK and P38. Furthermore, emodin could activate PPAR-γ and the anti-inflammatory effects of emodin can be reversed by GW9662, a specific antagonist for PPAR-γ. In conclusion, our results demonstrate that emodin activates PPAR-γ, thereby attenuating LPS-induced inflammatory response. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Inflammation, characterized by redness, swelling, heat, pain and dysfunction of the organs, is a fundamental protective response that sometimes goes awry and becomes a major cofactor in the pathogenesis of many diseases, such as mastitis [1,2]. Inflammation is usually caused by various factors such as microbial infection, tissue damage, and cardiac infarction. LPS, a potent virulence component from Gram negative bacteria, is a well-known inducer of the innate immune response and has been reported to be an important trigger of inflammation [3,4]. The innate immune system is the major contributor to acute inflammation induced by microbial infection or tissue damage [5,6]. Besides macrophages and dendritic cells (DCs), nonprofessional cells also play important roles such as epithelial cells, endothelial cells, and fibroblasts to innate immunity. Mammary epithelial cells (MEC) are the first line of the mammary gland in defensing the invading pathogens [7]. In vitro culture of MEC is widely used as a model to study the capacity of cells to sense and respond to mastitis-causing bacteria. Emodin (3-methyl-1, 6, 8-trihydroxyanthraquinone) is an anthraquinone derivative from the rhizome of Rheum palmatum that is widely used as a laxative in traditional Chinese medicine [8]. It has been reported that emodin shows a number of biological activities
⁎ Corresponding author at: Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun 130062, PR China. Tel./fax: + 86 431 87981351. E-mail address: [email protected] (X. Zhang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2014.05.019 1567-5769/© 2014 Elsevier B.V. All rights reserved.
such as immunosuppressive, hepatoprotective, anti-tumor and antiinflammatory activities [9–13]. Treatment of RAW 264.7 macrophages with emodin suppressed the expression of a panel of inflammatoryassociated genes, including tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), as well as the nuclear translocation of nuclear factor-κB (NF-κB) [14]. Recently, it has been reported to have a protective effect against lipopolysaccharide (LPS)-induced mastitis. However, the underlying molecular mechanisms are not well understood. Mammary epithelial cells have been reported to play an important role in defensing the invading pathogens. Thus, in the present study, LPS-stimulated mouse MEC was used as a tool to investigate the anti-inflammatory effect of emodin and elucidate the potential mechanisms. 2. Materials and methods 2.1. Materials Emodin (3-methyl-1, 6, 8-trihydroxyanthraquinone) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Jilin, China) (Fig. 1). LPS (Escherichia coli 055:B5) was purchased from Sigma (St. Louis, MO, USA). Dulbecco's modified eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen-Gibco (Grand Island, NY). Mouse TNF-α, IL-6 and IL-1β enzyme-linked immunosorbent assay (ELISA) kits were purchased from Biolegend (CA, USA). Rabbit monoclonal antibodies IκBα, p65, p-p65, ERK, p-ERK, p38, p-p38, JNK, p-JNK, iNOS, COX-2 and mouse monoclonal antibodies p-IκBα were purchased from Cell Signaling
Z. Yang et al. / International Immunopharmacology 21 (2014) 354–360
355
40 μg/ml for 18 h. Cell-free supernatants were collected for the proinflammatory cytokines assays with a mouse ELISA kit, according to the manufacturer's instructions (Biolegend, Inc, San Diego, CA, USA). 2.6. Western blot analysis
Fig. 1. Chemical structure of emodin.
Technology Inc (Beverly, MA, USA). Mouse mAb PPAR-γ was purchased from ABCAM. Horseradish peroxidase-conjugated goat anti-rabbit and goat-mouse antibodies were provided by GE Healthcare (Buckinghamshire, UK). All other chemicals were of reagent grade. 2.2. Animals BALB/c mice (8–12 day gravid), weighting approximately 20–24 g, were purchased from the Center of Experiment Animals of Baiqiuen Medical College of Jilin University (Jilin, China). All animal experiments were performed in accordance with the National Institutes of Health guide for the Care and Use of Laboratory Animal. All female and male mice were housed together respectively for 2–3 d to adapt themselves to the surroundings. Then 2 female and 1 male mice were randomly divided in each cage supplied with sufficient water and forages. All cages had been washed carefully and conducted with autoclave sterilization. The house had been sterilized thoroughly with disinfectants.
MEC (1 × 106) were seeded in 6-well plates. When the plate was confluent, MEC were pretreated with emodin for 1 h before LPS stimulation. After LPS stimulation for 1 h, the cells were collected and washed twice with ice-cold PBS. Total proteins from cells were extracted by M-PER Mammalian Protein Extraction Reagent (Thermo). Protein concentration was determined by the BCA method. Equal amounts of protein were loaded in each well and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which subsequently was transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked for 2 h with 5% skim milk in TBST on the shaker at room temperature and then washed 3 times for each time 10 min with Tri–Tween buffered saline (TTBS, 20 mM Tris–HCl buffer, pH 7.6, 137 mM NaCl and 0.05% Tween 20). The membrane was placed on Primary antibody diluted at a 1:1000 proportion in diluent buffer [5% (w/v) BSA and 0.1% Tween 20 in TBS] and incubated overnight at 4 °C on the shaker. Subsequently, the membrane was washed with PBS-T followed by incubation with the secondary antibody conjugated with horse-radish peroxidase at room temperature for 1 h. The membrane was again washed 3 times for 10 min each time as above and finally the results were generated by using an enhanced chemiluminescence (ECL) Western blotting kit. 2.7. PPAR-γ ligand binding assay
2.3. Culture of mouse MEC Primary cultured mMECs were prepared as previously described by Smalley. Briefly, the mammary glands were collected from BALB/c mice under sterile conditions in the middle of pregnancy. Then the mammary tissues were minced into pasty fine tissues and digested by collagenase I/II/trypsin mixture and shaken at 37 °C. After filtration to remove undissociated tissue and debris, the cells were collected by centrifugation at 250 g for 5 min three times. The cell pellets were resuspended in DMEM/F12 containing 10% FCS and incubated for 1 h at 37 °C, and then the supermatant was collected. This step was repeated three times until the fibroblasts were removed. After the last incubate, the cells were resuspended in DMEM/F12 containing 10% FCS, 0.5% transferrin, 0.1% T3, and 0.5% EGF, and were cultured at 37 °C. The culture media were changed once every 24 h. The mMECs were identified by cytokeratin-18 immunocytochemistry as described by our previously studies [15]. When the plate was confluent, MEC were incubated in the presence or absence of different concentrations of emodin at 1 h before LPS challenge.
The ligand binding activity was detected using a NuLigand PPAR-γ kit (Microsystems, Kyoto, Japan) according to the manufacturer's instruction. The NuLigand PPAR-γ kit is a cell free assay based on the ligand-dependent interaction between PPAR-γ and TIF-2, so called CoA–BAP system [16]. 2.8. PPAR-γ activation and inhibition To test the activation of PPAR-γ by emodin, the MEC were pretreated with emodin for 1 h before LPS incubation. After LPS (1 μg/ml)
2.4. Cell viability assay Cell viability was measured by MTT assay. The cells were plated at a density of 4 × 105 cells/ml in 96-well plates at 37 °C with 5% CO2 for 1 h and then treated with 50 μl emodin at different concentrations (0–40 μg/ml) for 1 h followed by stimulation with 50 μl LPS. 18 h of LPS stimulation later, 20 μl MTT (5 mg/ml) was added to each well and the cells were again incubated for 4 h. The supernatant was removed and the formation of formazan was resolved with 150 μl of DMSO each well. The optical density was measured at 570 nm on a microplate reader (TECAN, Austria). 2.5. ELISA assay 5
MEC were seeded in 24-well plates (10 cell/well) and incubated in the presence of either LPS 1 μg/ml alone or LPS plus emodin 10, 20, and
Fig. 2. Effect of emodin on the cell viability of mouse mammary epithelial cells. Cells were cultured with different concentrations of emodin (0–40 μg/ml) in the absence or presence of 1 mg/L LPS for 24 h. The cell viability was determined by MTT assay. The values presented are the means ± SEM of three independent experiments.
356
Z. Yang et al. / International Immunopharmacology 21 (2014) 354–360
Fig. 3. Emodin inhibits lipopolysaccharide (LPS)-induced cytokine production in a dose-dependent manner. Cells were treated with 1 μg/ml LPS in the absence or presence of emodin (10, 20, 40 μg/ml) for 24 h. Levels of TNF-α (A) and IL-6 (B) in culture supernatants were measured by ELISA. The data presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #p b 0.05 vs. control group; *p b 0.05, **p b 0.01 vs. LPS group.
stimulation for 9 h, the expression of PPAR-γ was detected by Western blotting. Meanwhile, to determine whether the anti-inflammatory effect of emodin is dependent on PPAR-γ activation, GW9662, the specific
PPAR-γ antagonist was added into the medium 30 min before emodin incubation. Then the cells were stimulated by LPS and NF-κB activation and the expression of inflammatory cytokines was detected.
Fig. 4. Emodin inhibits lipopolysaccharide (LPS)-induced NF-κB activation and IκBα degradation. Cells were preincubated with emodin (10, 20, 40 μg/ml) for 1 h and then treated with 1 μg/ml LPS for 1 h. Protein samples were analyzed by Western blot with specific antibodies. β-actin was used as a control. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #p b 0.05 vs. control group; *p b 0.05, **p b 0.01 vs. LPS group.
Z. Yang et al. / International Immunopharmacology 21 (2014) 354–360
2.9. Statistical analysis All data are expressed as mean ± S.E.M. The differences among the various experimental groups were analyzed by one-way ANOVA. P b 0.05 was considered to be statistically significant. 3. Results
357
anti-inflammatory effects of emodin in LPS-induced mouse MEC, the expressions of iNOS, COX-2, TNF-α and IL-6 were measured. The results showed that emodin alone did not induce the expressions of iNOS, COX-2, TNF-α and IL-6 compared with those in the control group. LPS caused a significant increase of the expressions of iNOS, COX-2, TNF-α and IL-6 compared with those in the control group (P b 0.01). Emodin markedly reduced the concentration of these cytokines at a dosedependent manner (Fig. 3).
3.1. Effects of emodin on cell viability To assess whether emodin exerted the potential cytotoxicity effect on mouse MEC, the cells were evaluated by the MTT assay after incubated for 18 h in the absence or presence of LPS. Our result displayed that emodin had no significant effects on cell viability at a dose-dependent concentrations (10, 20, 40 μg/ml) (Fig. 2). In other words, the viability of the cells was not nearly affected by the treatment with emodin. 3.2. Emodin decreased LPS-induced up-regulation of inflammatory mediators in LPS-induced mouse MEC TNF-α, IL-6, iNOS and COX-2 are major inflammatory mediators and play an important role in inflammatory diseases [17,18]. To evaluate the
3.3. Emodin blocked LPS-induced NF-κB pathway NF-κB is a multi-subunit nuclear transcription factor which rapidly activates the transcription of various cytokines and chemokines and plays a critical role in the inflammatory response [19,20]. In resting cells, NF-κB is bound to its inhibitor IκB in the cytoplasm. Once stimulated by LPS, IκB is phosphorylated and degraded, and then the released NF-κB is transferred into the nucleolus and up-regulates the transcription of the inflammatory factors [21]. To explore whether the antiinflammatory effects of emodin are mediated by NF-κB pathway in LPS-induced mouse MEC, the levels of NF-κB and IκBα were determined through Western blot. The results indicated that emodin significantly suppressed the activation of NF-κB and the degradation of IκBα (Fig. 4).
Fig. 5. Emodin inhibits lipopolysaccharide (LPS)-induced MAPK activation. Cells were preincubated with emodin (10, 20, 40 μg/ml) for 1 h and then treated with 1 μg/ml LPS for 1 h. Protein samples were analyzed by Western blot with specific antibodies. β-actin was used as a control. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #p b 0.05 vs. control group; *p b 0.05, **p b 0.01 vs. LPS group.
358
Z. Yang et al. / International Immunopharmacology 21 (2014) 354–360
3.4. Emodin inhibited LPS-induced MAPK pathway The mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in regulating gene expression in eukaryotic cells and links extracellular signals to the machinery that controls fundamental cellular processes such as growth, proliferation, differentiation, migration, and apoptosis [22,23]. To certify whether the MAPK pathway is involved in the anti-inflammatory effects of emodin, the levels of p38, JNK, and ERK MAPKs were detected through Western blot. The results demonstrated that emodin markedly inhibited the LPS-induced increase of phosphorylation of p38, ERK, and JNK in a dose-dependent manner (Fig. 5). 3.5. Emodin was a PPAR-γ ligand To test whether emodin served as a PPAR-γ ligand, we detected PPAR-γ ligand binding activity using the CoA–BAP system. As shown in Fig. 6, emodin showed a dose-dependent PPAR-γ ligand binding activity. It was suggested that emodin was a PPAR-γ ligand. 3.6. Effects of emodin on PPAR-γ activation Some studies have shown that some herbal products exert antiinflammatory effects by activating PPAR-γ [24–26]. In this study, we investigated the effects of emodin on PPAR-γ activation. The results showed that emodin up-regulated the expression of PPAR-γ in a dose manner (Fig. 7). 3.7. Effects of emodin on LPS-induce inflammatory response is PPAR-γ dependent As shown in Fig. 8, LPS increased the expression of TNF-α and IL-6 and emodin reduced TNF-α and IL-6 expression dramatically. Emodin inhibited NF-κB activation induced by LPS. Whereas, GW9662, the PPAR-γ specific antagonist could abolish the anti-inflammatory effect of emodin. 4. Discussion In the present study, the anti-inflammatory effects of emodin on LPS-stimulated mouse MEC were assessed. Our results showed that emodin clearly inhibited the expression of pro-inflammatory mediators
Fig. 6. PPAR-γ ligand binding activity of emodin. The binding affinity of emodin towards the PPAR-γ LBD at the indicated concentration was measured using a NuLigand PPAR-γ kit. The control group was expressed as ‘1’ and fold change was calculated as other groups vs control group. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. *p b 0.05, **p b 0.01 vs. control group.
Fig. 7. Effects of emodin on PPAR-γ expression in mouse mammary epithelial cells. Cells were pretreated with different concentrations of emodin (10, 20, 40 μg/ml) for 9 h. Protein samples were analyzed by western blot with specific antibodies. β-actin was used as a control. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. *p b 0.05, **p b 0.01 vs. control group.
and suppressed NF-κB and MAPK activation in LPS-stimulated mouse MEC. Furthermore, emodin could activate PPAR-γ and the antiinflammatory effects of emodin can be reversed by GW9662, a specific antagonist for PPAR-γ. It is suggested that emodin activates PPAR-γ, thereby attenuating LPS-induced inflammatory response. Inflammation is a protective response by the body to ensure removal of harmful stimuli as well as a healing process for repairing damaged tissue [27]. However, overproduction of pro-inflammatory cytokines by immune cells to overwhelm pathogens can be fatal. During infection, the inflammatory responses are regulated by the cytokine network [28]. It is well known that TNF-α and IL-6 involved in the initiation and development of acute inflammation are important inflammatory mediators released in response to various injurious stimuli. LPS, a potent stimuli, can arouse the production of TNF-α and IL-6 through activating host immune and inflammation cells. TNF-α is called primary cytokine, for it exerts an important role in initiating an acute inflammatory response [29]. TNF-α can induce infiltration and activation of neutrophils, impair vascular endothelial cells, up-regulate cellular adhesion molecules, promote the secretion of oxygen-derived free radicals and initiate and aggravate the cascade of other inflammatory mediators. IL-6 is a pleiotropic cytokine produced by the cells of immune and nonimmune origin. Increased production of IL-6 is associated with disturbances of homeostasis, such as trauma, sepsis, or inflammatory diseases [30]. Previous studies demonstrated that emodin could inhibit TNF-α and IL-6 in LPS-stimulated human umbilical vein endothelial cells [31]. In this study, our results showed that emodin markedly decreased the levels of TNF-α and IL-6 in a dose-dependent manner in LPS-stimulated mouse MEC. It suggested that emodin attenuated the inflammatory responses through inhibiting the production of pro-inflammatory cytokines. Studies have shown that the production of TNF-α and IL-6 is regulated by NF-κB and MAPKs. To further explore the anti-inflammatory mechanism of emodin, NF-κB and MAPK activation was detected. NF-κB is a ubiquitously expressed family of transcription factors controlling the expression of numerous genes involved in inflammatory and immune responses and cellular proliferation [32]. In resting
Z. Yang et al. / International Immunopharmacology 21 (2014) 354–360
359
Fig. 8. Effects of emodin on LPS-induced inflammatory response is PPAR-γ dependent. Cells were treated with 40 μg/ml emodin for 1 h, or 10 μM GW9662 for 30 min before emodin incubation, and stimulated with LPS for 24 h. Protein samples were analyzed by Western blot with NF-κB antibody. Levels of TNF-α (A) and IL-6 (B) in culture supernatants were measured by ELISA. The data presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #p b 0.05 vs. control group; *p b 0.05, **p b 0.01 vs. LPS group.
cells, NF-κB is sequestered in the cytoplasm by association with inhibitory proteins called IκBs. Once stimulated by LPS, IκB is then ubiquitinated and degraded by the proteasome, freeing NF-κB to be translocated into the nucleus and to exert its functions as transcription factor. Our data displayed that emodin obviously suppressed NF-κB activation and IκB degradation in LPS-stimulated mouse MEC. We also found that emodin significantly inhibited the phosphorylation of ERK, JNK, and p38 in LPS-induced mouse MEC. Taken together, our data revealed that emodin blocked the activation of NF-κB and MAPK signal pathways. Therefore, emodin may decrease the inflammatory response through blocking the activation of NF-κB and MAPK signal pathways. Peroxisome proliferator-activated receptors (PPARs) are ligandactivated transcription factors that play important roles in the regulation of a large number of biological processes including inflammation. Ligands for PPAR-γ have been reported to exert negative effects on inflammation and immune responses. Some studies have shown that some herbal products exert anti-inflammatory effects by activating PPAR-γ. Reports have shown that PPAR-γ agonists could negatively regulate LPS-induced inflammatory responses [33]. Thus, the effects of emodin on PPAR-γ expression were detected. In this study, we found that emodin could up-regulate the expression of PPAR-γ in a dose manner. We also found that the inhibitory effect of TNF-α and IL-6 by emodin can be reversed by GW9662, the PPAR-γ specific antagonist. Our results suggested that the ability of attenuating inflammation by emodin in mouse MEC was dependent on the PPAR-γ pathway. In summary, this study has demonstrated that emodin decreased the levels of the pro-inflammatory cytokines and suppressed NF-κB and MAPK signal pathways in LPS-induced mouse MEC. The promising anti-inflammatory mechanism of emodin may be that emodin activates PPAR-γ, thereby attenuating LPS-induced NF-κB and MAPK activation and the release of pro-inflammatory cytokines.
Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (Nos. 30972225 and 30771596).
References [1] Conese M, Assael BM. Bacterial infections and inflammation in the lungs of cystic fibrosis patients. Pediatr Infect Dis J 2001;20:207–13. [2] Ben-Neriah Y, Karin M. Inflammation meets cancer, with NF-[kappa] B as the matchmaker. Nat Immunol 2011;12:715–23. [3] Atabai K, Matthay MA. The pulmonary physician in critical care. 5: Acute lung injury and the acute respiratory distress syndrome: definitions and epidemiology. Thorax 2002;57:452–8. [4] Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, et al. Incidence and outcomes of acute lung injury. N Engl J Med 2005;353:1685–93. [5] Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006;124:783–801. [6] Beutler B, Jiang Z, Georgel P, Crozat K, Croker B, Rutschmann S, et al. Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large. Annu Rev Immunol 2006;24:353–89. [7] Strandberg Y, Gray C, Vuocolo T, Donaldson L, Broadway M, Tellam R. Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells. Cytokine 2005;31:72–86. [8] Shi YQ, Fukai T, Sakagami H, Kuroda J, Miyaoka R, Tamura M, et al. Cytotoxic and DNA damage-inducing activities of low molecular weight phenols from rhubarb. Anticancer Res 2001;21:2847–53. [9] Huang HC, Lee CR, Chao PD, Chen CC, Chu SH. Vasorelaxant effect of emodin, an anthraquinone from a Chinese herb. Eur J Pharmacol 1991;205:289–94. [10] Kuo YC, Meng HC, Tsai WJ. Regulation of cell proliferation, inflammatory cytokine production and calcium mobilization in primary human T lymphocytes by emodin from Polygonum hypoleucum Ohwi. Inflamm Res 2001;50:73–82. [11] Lin CC, Chang CH, Yang JJ, Namba T, Hattori M. Hepatoprotective effects of emodin from Ventilago leiocarpa. J Ethnopharmacol 1996;52:107–11. [12] Chang CJ, Ashendel CL, Geahlen RL, McLaughlin JL, Waters DJ. Oncogene signal transduction inhibitors from medicinal plants. In Vivo 1996;10:185–90. [13] Huang Z, Chen G, Shi P. Effects of emodin on the gene expression profiling of human breast carcinoma cells. Cancer Detect Prev 2009;32:286–91. [14] Li HL, Chen HL, Li H, Zhang KL, Chen XY, Wang XW, et al. Regulatory effects of emodin on NF-kappaB activation and inflammatory cytokine expression in RAW 264.7 macrophages. Int J Mol Med 2005;16:41–7. [15] Fu Y, Zhou E, Wei Z, Liang D, Wang W, Wang T, et al. Glycyrrhizin inhibits the inflammatory response in mouse mammary epithelial cells and mouse mastitis model. FEBS J 2014. [16] Kanayama T, Mamiya S, Nishihara T, Nishikawa J. Basis of a high-throughput method for nuclear receptor ligands. J Biochem 2003;133:791–7. [17] Satoh H, Firestein GS, Billings PB, Harris JP, Keithley EM. Proinflammatory cytokine expression in the endolymphatic sac during inner ear inflammation. J Assoc Res Otolaryngol 2003;4:139–47. [18] Libby P. Coronary artery injury and the biology of atherosclerosis: inflammation, thrombosis, and stabilization. Am J Cardiol 2000;86:3J–8J (discussion J-9J). [19] Caamano J, Hunter CA. NF-kappaB family of transcription factors: central regulators of innate and adaptive immune functions. Clin Microbiol Rev 2002;15:414–29.
360
Z. Yang et al. / International Immunopharmacology 21 (2014) 354–360
[20] in Csl-iimbinf-kBa, in RmCsl-iimbinf-kBa, RAW 264.7 macrophagesVallabhapurapu S, Karin M. Regulation and function of NF-kappaB transcription factors in the immune system. Annu Rev Immunol 2009;27:693–733. [21] Choi YH, Jin GY, Li GZ, Yan GH. Cornuside suppresses lipopolysaccharide-induced inflammatory mediators by inhibiting nuclear factor-kappa B activation in RAW 264.7 macrophages. Biol Pharm Bull 2011;34:959–66. [22] Mordret G. MAP kinase kinase: a node connecting multiple pathways. Biol Cell 1993;79:193–207. [23] Jebamercy G, Vigneshwari L, Balamurugan K. A MAP Kinase pathway in Caenorhabditis elegans is required for defense against infection by opportunistic Proteus species. Microbes Infect 2013;15:550–68. [24] Lim HA, Lee EK, Kim JM, Park MH, Kim DH, Choi YJ, et al. PPARgamma activation by baicalin suppresses NF-kappaB-mediated inflammation in aged rat kidney. Biogerontology 2012;13:133–45. [25] Park MY, Lee KS, Sung MK. Effects of dietary mulberry, Korean red ginseng, and banaba on glucose homeostasis in relation to PPAR-alpha, PPAR-gamma, and LPL mRNA expressions. Life Sci 2005;77:3344–54. [26] Song MK, Roufogalis BD, Huang TH. Modulation of diabetic retinopathy pathophysiology by natural medicines through PPAR-gamma-related pharmacology. Br J Pharmacol 2012;165:4–19.
[27] Medzhitov R. Origin and physiological roles of inflammation. Nature 2008;454:428–35. [28] Lee JW, Bannerman DD, Paape MJ, Huang MK, Zhao X. Characterization of cytokine expression in milk somatic cells during intramammary infections with Escherichia coli or Staphylococcus aureus by real-time PCR. Vet Res 2006;37:219–29. [29] Skotnicka B, Hassmann E. Proinflammatory and immunoregulatory cytokines in the middle ear effusions. Int J Pediatr Otorhinolaryngol 2008;72:13–7. [30] Kishimoto T, Akira S, Taga T. Interleukin-6 and its receptor: a paradigm for cytokines. Science 1992;258:593–7. [31] Meng G, Liu Y, Lou C, Yang H. Emodin suppresses lipopolysaccharide-induced proinflammatory responses and NF-kappaB activation by disrupting lipid rafts in CD14-negative endothelial cells. Br J Pharmacol 2010;161:1628–44. [32] Ghosh S, Karin M. Missing pieces in the NF-kappaB puzzle. Cell 2002;109(Suppl.): S81–96. [33] Li H, Ruan XZ, Powis SH, Fernando R, Mon WY, Wheeler DC, et al. EPA and DHA reduce LPS-induced inflammation responses in HK-2 cells: evidence for a PPAR-gamma-dependent mechanism. Kidney Int 2005;67:867–74.
