Cellular Physiology and Biochemistry
Cell Physiol Biochem 2014;34:590-602 DOI: 10.1159/000363025 Published online: August 11, 2014
© 2014 S. Karger AG, Basel www.karger.com/cpb
Xie et al.: GLP-2 Inhibits the Inflammation Accepted: June 10, 2014 ERK and NF-kB Pathway to Suppress 1421-9778/14/0342-0590$39.50/0 This is an Open Access article licensed under the terms of the Creative Commons AttributionNonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only.
Original Paper
GLP-2 Suppresses LPS-Induced Inflammation in Macrophages by Inhibiting ERK Phosphorylation and NF-κB Activation Shanshan Xiea Bingrun Liua Shoupeng Fub Wei Wangb Yunhou Yinc Nan Lia Wei Chenb Juxiong Liub Dianfeng Liua College of Animal Science (Laboratory Animal Center), Jilin University, bCollege of Veterinary Medicine, Jilin University, Changchun, cGuizhou Minzu University, Guiyang, P.R. China a
Key Words GLP-2 • Pro-inflammatory cytokines • ERK • NF-κB Abstract Background/Aims: GLP-2 has been shown to exert anti-inflammatory effects, but the underlying molecular mechanisms remained undefined. As macrophages are important in the development and maintenance of inflammation, we investigated whether exogenous GLP-2 modulates the expression of pro-inflammatory proteins in LPS stimulated murine peritoneal macrophages. Methods: Macrophages were pretreated with various concentrations of GLP-2 for 1 h and then stimulated with LPS. The effects on pro-inflammatory enzymes (iNOS and COX-2), and pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) were analysed by Western blotting, ELISA and qRT-PCR. We also examined whether NF-κB or MAPK signaling was involved in the effects of GLP-2. Results: In macrophages, GLP-2 blunted the effect of LPS on protein and mRNA expression levels of iNOS, COX-2, TNF-α, IL-1β and IL-6. Pre-incubation of macrophages with GLP-2 also blunted LPS-induced IκB-α degradation, IκB-α phosphorylation and NF-κB translocation. In the presence of GLP-2, the effect of LPS treatment on ERK phosphorylation was also profoundly blunted. GLP-2 did, however, not significantly modify the effects of LPS on p38 and JNK activities. Conclusions: These findings demonstrate that in LPS primed macrophages, GLP-2 reduced pro-inflammatory enzymes and cytokine production via mechanisms involving the suppression of NF-κB activity and ERK phosphorylation. Copyright © 2014 S. Karger AG, Basel
S. Xie, B. Liu, S. Fu, W. Wang and Y. Yin contributed equally. Juxiong Liu and Dianfeng Liu
College of Animal Science (Laboratory Animal Center) Jilin University 5333 Xi’an Road, Jilin 130062 (China) Tel. +86-431-87836163, E-Mail [email protected] and E-Mail [email protected]
590
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2014;34:590-602 DOI: 10.1159/000363025 Published online: August 11, 2014
© 2014 S. Karger AG, Basel www.karger.com/cpb
Xie et al.: GLP-2 Inhibits ERK and NF-kB Pathway to Suppress the Inflammation
Introduction
Macrophages are highly heterogeneous hematopoietic cells found in nearly every tissue in the body [1]. Canonically, these cells have been defined as the sentinels of the innate immune system, monitoring the varied tissue milieu for early signs of infection or tissue damage. Despite the daunting array of inputs, macrophage responses are coordinated through two distinct and mutually exclusive activation programs, termed classical (or M1) and alternative (or M2) [2, 3]. These activation programs were initially defined by their antimicrobial activities. Classical activation occurs in response to products derived from or associated with bacterial infections, such as lipopolysaccharide (LPS) and interferon γ (IFN-γ), and results in highly inflammatory macrophages with high phagocytic and bactericidal potential [2, 4]. In contrast, alternative activation occurs in response to products derived from or associated with parasitic infections, such as Schistosoma egg antigen, interleukin-4 (IL-4) and interleukin-13 (IL-13), and promotes anti-parasitic functionalities tissue repair and remodeling [5]. Microbial antigens lead to the classical activated macrophages (M1) and the consequent release of pro-inflammatory and/or cytotoxic factors such as tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in tissues. These pro-inflammatory cytokines and mediators released from macrophages are vital to the regulation of the immune response, but the dysregulation of their production can lead to pathological conditions, such as septic shock, rheumatoid arthritis and other chronic inflammatory diseases [6]. LPS is a bacterial endotoxin used to study experimentally induced inflammation. Mechanistically, LPS stimulates toll-like receptor 4 (TLR4) in various cells, including macrophages, to activate nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs), which are classified into at least three components: extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK) and p38 MAPK [7]. MAPKs have been implicated in the release of immune-related cytotoxic factors, such as iNOS, COX-2 and proinflammatory cytokines (TNF-α, IL-1β, IL-6) [8, 9]. Glucagon-like peptide 2 (GLP-2), a 33-amino acid peptide (GLP-2 (1-33)), belongs to the so-called proglucagon-derived peptides (PGDPs), which originate from tissue-specific post-translational processing by convertases of proglucagon. GLP-2 (1-33) is synthesized in the gastrointestinal tract in the L-cells of the small intestine and colon [10, 11]. Studies in rodents treated with GLP-2 (1-33) or synthetic analogs of the peptide demonstrate that GLP2 promotes the growth of the small intestine [12-14], improves intestinal wound healing through in a transforming growth factor β (TGF-β)-mediated process [15], decreases mortality in indomethacin-induced murine enteritis and enhances enteric adaptation in rodent shortbowel models [16-19]. In addition to these intestinal trophic effects, other studies have demonstrated that GLP-2 attenuates inflammation [20]. However, the underlying molecular mechanisms have not been resolved. The present study attempted to elucidate the anti-inflammatory potential of GLP2 on the inflammatory response induced by LPS in murine peritoneal macrophages. The involvement of IκB-α, NF-κB and MAPKs was also examined to further investigate the underlying mechanisms. We showed that LPS-induced pro-inflammatory enzymes and proinflammatory cytokines in peritoneal macrophages were significantly reduced by treatment with GLP-2 and the inhibitory effect of GLP-2 on pro-inflammatory enzymes and proinflammatory cytokines was mediated by ERK phosphorylation and NF-κB signaling. Materials and Methods
Animals Balb/c female mice (6-8 weeks old, 17-21 g) were obtained from the Center of Experimental Animal of Baiqiuen Medical College of Jilin University (Jilin, China) and maintained in plastic cages under conventional conditions. Water and pellet diets were supplied ad libitum. Studies were performed in accordance with the guidelines established by the Jilin University Institutional Animal Care and Use Committee.
591
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2014;34:590-602 DOI: 10.1159/000363025 Published online: August 11, 2014
© 2014 S. Karger AG, Basel www.karger.com/cpb
Xie et al.: GLP-2 Inhibits ERK and NF-kB Pathway to Suppress the Inflammation
Table 1. The primer sequences of β-actin, GLP-2R, iNOS, COX-2, TNF-α, IL-1β and IL-6
Peritoneal macrophages cultures and treatments Peritoneal exudates were obtained from Balb/c female mice by lavage 4 days after an intraperitoneal injection of 4 ml sterile 4% thioglycollate broth. After washing with RPMI 1640 medium, the cell suspensions were centrifuged at 800 g at 4 °C for 5 min. The red blood cells were eliminated by ACK buffer and the cells were washed and resuspended in RPMI 1640 supplemented with 10% inactivated FBS, 10 mM HEPES, 2 mM glutamine, and 100 U/ml penicillin-100 mg/ml streptomycin. Peritoneal macrophages were plated in 6-cm tissue culture dishes at 37 °C in a 5% CO2 humidified atmosphere. Changes in cell morphology and growth conditions were carefully monitored using an inverted microscope. Macrophages were precultured in serum-free RPMI 1640 medium for 24 h to reduce mitogenic effects. Macrophages were pretreated with various concentrations of GLP-2 for 1 h and stimulated with LPS.
Cell viability assay Cell viability was determined by MTT assay. Briefly, macrophages were seeded into 96-well plates at a density of 2×104 cells per well 24 h before treatment. Cells were treated with various concentrations of GLP-2 for the indicated time periods followed by incubating with 5 mg/ml of MTT working solution for 4 h at 37 °C. After added 100 μl of DMSO to dissolve the crystals, the absorbance of each well at 570 nm was measured using a Synergy 2 Multi-Mode Microplate Reader (BIO-TEK, INC). Three replicates were carried out for each of the different treatments. RNA extraction, reverse transcriptase PCR and quantitative real-time PCR analysis Total RNA was extracted from peritoneal macrophages using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the supplier’s protocol. Complementary DNA (cDNA) was generated from 5 μg of total RNA using PrimeScript RT reagent Kit with gDNA Eraser (Takara Shuzo Co, Ltd, Kyoto, Japan). The mRNA levels of various genes were evaluated by quantitative polymerase chain reaction (qRT-PCR) analysis and the SYBR Green QuantiTect RT-PCR Kit (Roche, South San Francisco, CA, USA), performed in triplicate for each sample. The relative expression levels of iNOS, COX-2, TNF-α, IL-1β and IL-6 were calculated relative to β-actin using the comparative cycle threshold method. The primer sequences for the tested genes are shown in Table 1.
ELISA Macrophages seeded in 24-well plates were pretreated with various concentrations of GLP-2 for 1 h followed by stimulation with LPS (100 ng/ml) for another 12 h. After stimulation, the culture media were collected and centrifuged at 13,000 g for 3 min. The levels of cytokines in the supernatants for TNF-α, IL1β and IL-6 were determined by ELISA (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. Three replicates were performed for each of the different treatments. Western blot analysis Cells were harvested with ice-cold PBS and centrifuged at 13,000×g for 3 min at 4 °C. Nuclear and cytosolic extracts were prepared using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute
592
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2014;34:590-602 DOI: 10.1159/000363025 Published online: August 11, 2014
© 2014 S. Karger AG, Basel www.karger.com/cpb
Xie et al.: GLP-2 Inhibits ERK and NF-kB Pathway to Suppress the Inflammation
of Biotechnology, Jiangsu, China) according to the manufacturer’s instructions. Protein concentrations were measured using a bicinchoninic acid protein assay kit (Beyotime Co, China). Equal amounts of lysates (50 μg) were separated on 10% SDS-PAGE. Proteins were transferred onto immunoblot polyvinylidene difluoride membranes (Chemicon International, Millipore, Billerica, MA), and the membranes were blocked with 5% BSA in Tris-buffered saline with 0.1% Tween (TBS-T) for 2 h and incubated overnight at 4 °C with the following primary antibodies: iNOS (1:2000), COX-2 (1:1000) (Abcam, Cambridge, CA, USA), phosphoERK1/2 (1:1000), ERK1/2 (1:1000), phospho-p38 (1:1000), p38 (1:1000), phospho-JNK (1:1000), JNK (1:1000), IκB-α (1:1000), phospho-IκB-α (1:1000) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-mouse NF-κB/RelA (1:1000; Santa Cruz Biotechnology), PCNA (1:1000; Santa Cruz Biotechnology), or β-actin (1:2000; Santa Cruz Biotechnology). Blots were washed four times for 15 min each in TBS-T and incubated with horseradish peroxidase-labeled secondary goat anti-rabbit (1:2000; Santa Cruz Biotechnology) or rabbit anti-goat (1:2000; Santa Cruz Biotechnology) for 1 h. Blots were again washed four times for 15 min each in TBS-T. Finally, blots were developed using the enhanced chemiluminescence (Pplygen Co, China) method. Statistical analyses The results are expressed as the means ± SD. Data were analyzed using the statistical software package SPSS 12.0(SPSS Inc, Chicago, IL, USA). Groups were compared by one-way analysis of variance (ANOVA) followed by the least significant difference test. A P value of less than 0.05 was considered statistically significant, and values less than 0.01 were considered markedly significant.
Results
GLP-2 inhibits LPS-stimulated expression of iNOS and COX-2 proteins and mRNA in macrophages iNOS and COX-2 are two important pro-inflammatory proteins correlated with LPS stimulation in macrophages. Macrophages were pretreated with GLP-2 (10-9, 10-8, 10-7 and 10-6 M) for 1 h and stimulated with LPS (100 ng/ml) for 4 h to investigate the effect of GLP-2 on the activation of LPS-stimulated macrophages. iNOS and COX-2 were examined by Western blotting and qRT-PCR. GLP-2 notably dose-dependently inhibited the increased expression of iNOS and COX-2 proteins and mRNA stimulated by LPS (Fig. 1). GLP-2 attenuates LPS-stimulated the production of the pro-inflammatory cytokines TNF-a, IL-1β and IL-6 at the transcriptional and translational levels in macrophages Pro-inflammatory cytokines (including TNF-α, IL-1β and IL-6) play important roles in the inflammatory process. Macrophages were stimulated with LPS (100 ng/ml) in the presence or absence of GLP-2 (10-9, 10-8, 10-7 and 10-6 M) to investigate whether GLP-2 represses the production of these pro-inflammatory cytokines. Fig. 2 shows that the significant increases in the proteins and mRNA levels of TNF-α (Figs. 2A, 2B), IL-1β (Fig. 2E) and IL-6 (Figs. 2C, 2D) resulting from the LPS stimulation were inhibited by GLP-2 in a dose-dependent manner in macrophages. The secretion of IL-1β was not detected in cell culture supernatants. We investigated the dose effect of GLP-2 on cell viability by the MTT assay to determine the GLP-2 cytotoxicity. Macrophages were incubated with various doses of GLP-2 for 24 h. The MTT assay showed that GLP-2 did not affect cell viability even at a high concentration of 10-6 M (Fig. 2F), which demonstrates that GLP-2 at non-cytotoxic levels suppressed LPSinduced inflammatory responses in macrophages via attenuation of iNOS, COX-2 and proinflammatory cytokines expression in our experiments. Effects of GLP-2 are not mediated by the GLP-2 receptor The GLP-2 receptor (GLP-2R) is the functional receptor of GLP-2. We investigated whether GLP-2R mRNA was expressed in macrophages to analyze whether this receptor mediates anti-inflammation of GLP-2. Fig. 3 shows that GLP-2R mRNA was not detected in macrophages. Therefore, GLP-2 can not act on macrophages via the GLP-2 receptor.
593
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2014;34:590-602 DOI: 10.1159/000363025 Published online: August 11, 2014
© 2014 S. Karger AG, Basel www.karger.com/cpb
Xie et al.: GLP-2 Inhibits ERK and NF-kB Pathway to Suppress the Inflammation
Fig. 1. Effects of GLP-2 on LPS-induced expression of proteins and mRNA of iNOS and COX-2 in macrophages. Macrophages were pretreated with GLP-2 (10-9, 10-8, 10-7 and 10-6 M) 1 h prior to incubation of LPS (100 ng/mL) for 4 h. Proteins and mRNA of iNOS and COX-2 were determined by Western blotting and qRT-PCR. Panels A and B show mRNA of iNOS (A) and COX-2 (B). Panels C, D and E show the protein expression of iNOS (C, D) and COX-2 (C, E), with levels normalized to β-actin. The results are expressed as the means ± SD for each group from three independent experiments. # Significant compared to control alone, p
Cell Physiol Biochem 2014;34:590-602 DOI: 10.1159/000363025 Published online: August 11, 2014
© 2014 S. Karger AG, Basel www.karger.com/cpb
Xie et al.: GLP-2 Inhibits the Inflammation Accepted: June 10, 2014 ERK and NF-kB Pathway to Suppress 1421-9778/14/0342-0590$39.50/0 This is an Open Access article licensed under the terms of the Creative Commons AttributionNonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only.
Original Paper
GLP-2 Suppresses LPS-Induced Inflammation in Macrophages by Inhibiting ERK Phosphorylation and NF-κB Activation Shanshan Xiea Bingrun Liua Shoupeng Fub Wei Wangb Yunhou Yinc Nan Lia Wei Chenb Juxiong Liub Dianfeng Liua College of Animal Science (Laboratory Animal Center), Jilin University, bCollege of Veterinary Medicine, Jilin University, Changchun, cGuizhou Minzu University, Guiyang, P.R. China a
Key Words GLP-2 • Pro-inflammatory cytokines • ERK • NF-κB Abstract Background/Aims: GLP-2 has been shown to exert anti-inflammatory effects, but the underlying molecular mechanisms remained undefined. As macrophages are important in the development and maintenance of inflammation, we investigated whether exogenous GLP-2 modulates the expression of pro-inflammatory proteins in LPS stimulated murine peritoneal macrophages. Methods: Macrophages were pretreated with various concentrations of GLP-2 for 1 h and then stimulated with LPS. The effects on pro-inflammatory enzymes (iNOS and COX-2), and pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) were analysed by Western blotting, ELISA and qRT-PCR. We also examined whether NF-κB or MAPK signaling was involved in the effects of GLP-2. Results: In macrophages, GLP-2 blunted the effect of LPS on protein and mRNA expression levels of iNOS, COX-2, TNF-α, IL-1β and IL-6. Pre-incubation of macrophages with GLP-2 also blunted LPS-induced IκB-α degradation, IκB-α phosphorylation and NF-κB translocation. In the presence of GLP-2, the effect of LPS treatment on ERK phosphorylation was also profoundly blunted. GLP-2 did, however, not significantly modify the effects of LPS on p38 and JNK activities. Conclusions: These findings demonstrate that in LPS primed macrophages, GLP-2 reduced pro-inflammatory enzymes and cytokine production via mechanisms involving the suppression of NF-κB activity and ERK phosphorylation. Copyright © 2014 S. Karger AG, Basel
S. Xie, B. Liu, S. Fu, W. Wang and Y. Yin contributed equally. Juxiong Liu and Dianfeng Liu
College of Animal Science (Laboratory Animal Center) Jilin University 5333 Xi’an Road, Jilin 130062 (China) Tel. +86-431-87836163, E-Mail [email protected] and E-Mail [email protected]
590
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2014;34:590-602 DOI: 10.1159/000363025 Published online: August 11, 2014
© 2014 S. Karger AG, Basel www.karger.com/cpb
Xie et al.: GLP-2 Inhibits ERK and NF-kB Pathway to Suppress the Inflammation
Introduction
Macrophages are highly heterogeneous hematopoietic cells found in nearly every tissue in the body [1]. Canonically, these cells have been defined as the sentinels of the innate immune system, monitoring the varied tissue milieu for early signs of infection or tissue damage. Despite the daunting array of inputs, macrophage responses are coordinated through two distinct and mutually exclusive activation programs, termed classical (or M1) and alternative (or M2) [2, 3]. These activation programs were initially defined by their antimicrobial activities. Classical activation occurs in response to products derived from or associated with bacterial infections, such as lipopolysaccharide (LPS) and interferon γ (IFN-γ), and results in highly inflammatory macrophages with high phagocytic and bactericidal potential [2, 4]. In contrast, alternative activation occurs in response to products derived from or associated with parasitic infections, such as Schistosoma egg antigen, interleukin-4 (IL-4) and interleukin-13 (IL-13), and promotes anti-parasitic functionalities tissue repair and remodeling [5]. Microbial antigens lead to the classical activated macrophages (M1) and the consequent release of pro-inflammatory and/or cytotoxic factors such as tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in tissues. These pro-inflammatory cytokines and mediators released from macrophages are vital to the regulation of the immune response, but the dysregulation of their production can lead to pathological conditions, such as septic shock, rheumatoid arthritis and other chronic inflammatory diseases [6]. LPS is a bacterial endotoxin used to study experimentally induced inflammation. Mechanistically, LPS stimulates toll-like receptor 4 (TLR4) in various cells, including macrophages, to activate nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs), which are classified into at least three components: extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK) and p38 MAPK [7]. MAPKs have been implicated in the release of immune-related cytotoxic factors, such as iNOS, COX-2 and proinflammatory cytokines (TNF-α, IL-1β, IL-6) [8, 9]. Glucagon-like peptide 2 (GLP-2), a 33-amino acid peptide (GLP-2 (1-33)), belongs to the so-called proglucagon-derived peptides (PGDPs), which originate from tissue-specific post-translational processing by convertases of proglucagon. GLP-2 (1-33) is synthesized in the gastrointestinal tract in the L-cells of the small intestine and colon [10, 11]. Studies in rodents treated with GLP-2 (1-33) or synthetic analogs of the peptide demonstrate that GLP2 promotes the growth of the small intestine [12-14], improves intestinal wound healing through in a transforming growth factor β (TGF-β)-mediated process [15], decreases mortality in indomethacin-induced murine enteritis and enhances enteric adaptation in rodent shortbowel models [16-19]. In addition to these intestinal trophic effects, other studies have demonstrated that GLP-2 attenuates inflammation [20]. However, the underlying molecular mechanisms have not been resolved. The present study attempted to elucidate the anti-inflammatory potential of GLP2 on the inflammatory response induced by LPS in murine peritoneal macrophages. The involvement of IκB-α, NF-κB and MAPKs was also examined to further investigate the underlying mechanisms. We showed that LPS-induced pro-inflammatory enzymes and proinflammatory cytokines in peritoneal macrophages were significantly reduced by treatment with GLP-2 and the inhibitory effect of GLP-2 on pro-inflammatory enzymes and proinflammatory cytokines was mediated by ERK phosphorylation and NF-κB signaling. Materials and Methods
Animals Balb/c female mice (6-8 weeks old, 17-21 g) were obtained from the Center of Experimental Animal of Baiqiuen Medical College of Jilin University (Jilin, China) and maintained in plastic cages under conventional conditions. Water and pellet diets were supplied ad libitum. Studies were performed in accordance with the guidelines established by the Jilin University Institutional Animal Care and Use Committee.
591
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2014;34:590-602 DOI: 10.1159/000363025 Published online: August 11, 2014
© 2014 S. Karger AG, Basel www.karger.com/cpb
Xie et al.: GLP-2 Inhibits ERK and NF-kB Pathway to Suppress the Inflammation
Table 1. The primer sequences of β-actin, GLP-2R, iNOS, COX-2, TNF-α, IL-1β and IL-6
Peritoneal macrophages cultures and treatments Peritoneal exudates were obtained from Balb/c female mice by lavage 4 days after an intraperitoneal injection of 4 ml sterile 4% thioglycollate broth. After washing with RPMI 1640 medium, the cell suspensions were centrifuged at 800 g at 4 °C for 5 min. The red blood cells were eliminated by ACK buffer and the cells were washed and resuspended in RPMI 1640 supplemented with 10% inactivated FBS, 10 mM HEPES, 2 mM glutamine, and 100 U/ml penicillin-100 mg/ml streptomycin. Peritoneal macrophages were plated in 6-cm tissue culture dishes at 37 °C in a 5% CO2 humidified atmosphere. Changes in cell morphology and growth conditions were carefully monitored using an inverted microscope. Macrophages were precultured in serum-free RPMI 1640 medium for 24 h to reduce mitogenic effects. Macrophages were pretreated with various concentrations of GLP-2 for 1 h and stimulated with LPS.
Cell viability assay Cell viability was determined by MTT assay. Briefly, macrophages were seeded into 96-well plates at a density of 2×104 cells per well 24 h before treatment. Cells were treated with various concentrations of GLP-2 for the indicated time periods followed by incubating with 5 mg/ml of MTT working solution for 4 h at 37 °C. After added 100 μl of DMSO to dissolve the crystals, the absorbance of each well at 570 nm was measured using a Synergy 2 Multi-Mode Microplate Reader (BIO-TEK, INC). Three replicates were carried out for each of the different treatments. RNA extraction, reverse transcriptase PCR and quantitative real-time PCR analysis Total RNA was extracted from peritoneal macrophages using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the supplier’s protocol. Complementary DNA (cDNA) was generated from 5 μg of total RNA using PrimeScript RT reagent Kit with gDNA Eraser (Takara Shuzo Co, Ltd, Kyoto, Japan). The mRNA levels of various genes were evaluated by quantitative polymerase chain reaction (qRT-PCR) analysis and the SYBR Green QuantiTect RT-PCR Kit (Roche, South San Francisco, CA, USA), performed in triplicate for each sample. The relative expression levels of iNOS, COX-2, TNF-α, IL-1β and IL-6 were calculated relative to β-actin using the comparative cycle threshold method. The primer sequences for the tested genes are shown in Table 1.
ELISA Macrophages seeded in 24-well plates were pretreated with various concentrations of GLP-2 for 1 h followed by stimulation with LPS (100 ng/ml) for another 12 h. After stimulation, the culture media were collected and centrifuged at 13,000 g for 3 min. The levels of cytokines in the supernatants for TNF-α, IL1β and IL-6 were determined by ELISA (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. Three replicates were performed for each of the different treatments. Western blot analysis Cells were harvested with ice-cold PBS and centrifuged at 13,000×g for 3 min at 4 °C. Nuclear and cytosolic extracts were prepared using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute
592
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2014;34:590-602 DOI: 10.1159/000363025 Published online: August 11, 2014
© 2014 S. Karger AG, Basel www.karger.com/cpb
Xie et al.: GLP-2 Inhibits ERK and NF-kB Pathway to Suppress the Inflammation
of Biotechnology, Jiangsu, China) according to the manufacturer’s instructions. Protein concentrations were measured using a bicinchoninic acid protein assay kit (Beyotime Co, China). Equal amounts of lysates (50 μg) were separated on 10% SDS-PAGE. Proteins were transferred onto immunoblot polyvinylidene difluoride membranes (Chemicon International, Millipore, Billerica, MA), and the membranes were blocked with 5% BSA in Tris-buffered saline with 0.1% Tween (TBS-T) for 2 h and incubated overnight at 4 °C with the following primary antibodies: iNOS (1:2000), COX-2 (1:1000) (Abcam, Cambridge, CA, USA), phosphoERK1/2 (1:1000), ERK1/2 (1:1000), phospho-p38 (1:1000), p38 (1:1000), phospho-JNK (1:1000), JNK (1:1000), IκB-α (1:1000), phospho-IκB-α (1:1000) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-mouse NF-κB/RelA (1:1000; Santa Cruz Biotechnology), PCNA (1:1000; Santa Cruz Biotechnology), or β-actin (1:2000; Santa Cruz Biotechnology). Blots were washed four times for 15 min each in TBS-T and incubated with horseradish peroxidase-labeled secondary goat anti-rabbit (1:2000; Santa Cruz Biotechnology) or rabbit anti-goat (1:2000; Santa Cruz Biotechnology) for 1 h. Blots were again washed four times for 15 min each in TBS-T. Finally, blots were developed using the enhanced chemiluminescence (Pplygen Co, China) method. Statistical analyses The results are expressed as the means ± SD. Data were analyzed using the statistical software package SPSS 12.0(SPSS Inc, Chicago, IL, USA). Groups were compared by one-way analysis of variance (ANOVA) followed by the least significant difference test. A P value of less than 0.05 was considered statistically significant, and values less than 0.01 were considered markedly significant.
Results
GLP-2 inhibits LPS-stimulated expression of iNOS and COX-2 proteins and mRNA in macrophages iNOS and COX-2 are two important pro-inflammatory proteins correlated with LPS stimulation in macrophages. Macrophages were pretreated with GLP-2 (10-9, 10-8, 10-7 and 10-6 M) for 1 h and stimulated with LPS (100 ng/ml) for 4 h to investigate the effect of GLP-2 on the activation of LPS-stimulated macrophages. iNOS and COX-2 were examined by Western blotting and qRT-PCR. GLP-2 notably dose-dependently inhibited the increased expression of iNOS and COX-2 proteins and mRNA stimulated by LPS (Fig. 1). GLP-2 attenuates LPS-stimulated the production of the pro-inflammatory cytokines TNF-a, IL-1β and IL-6 at the transcriptional and translational levels in macrophages Pro-inflammatory cytokines (including TNF-α, IL-1β and IL-6) play important roles in the inflammatory process. Macrophages were stimulated with LPS (100 ng/ml) in the presence or absence of GLP-2 (10-9, 10-8, 10-7 and 10-6 M) to investigate whether GLP-2 represses the production of these pro-inflammatory cytokines. Fig. 2 shows that the significant increases in the proteins and mRNA levels of TNF-α (Figs. 2A, 2B), IL-1β (Fig. 2E) and IL-6 (Figs. 2C, 2D) resulting from the LPS stimulation were inhibited by GLP-2 in a dose-dependent manner in macrophages. The secretion of IL-1β was not detected in cell culture supernatants. We investigated the dose effect of GLP-2 on cell viability by the MTT assay to determine the GLP-2 cytotoxicity. Macrophages were incubated with various doses of GLP-2 for 24 h. The MTT assay showed that GLP-2 did not affect cell viability even at a high concentration of 10-6 M (Fig. 2F), which demonstrates that GLP-2 at non-cytotoxic levels suppressed LPSinduced inflammatory responses in macrophages via attenuation of iNOS, COX-2 and proinflammatory cytokines expression in our experiments. Effects of GLP-2 are not mediated by the GLP-2 receptor The GLP-2 receptor (GLP-2R) is the functional receptor of GLP-2. We investigated whether GLP-2R mRNA was expressed in macrophages to analyze whether this receptor mediates anti-inflammation of GLP-2. Fig. 3 shows that GLP-2R mRNA was not detected in macrophages. Therefore, GLP-2 can not act on macrophages via the GLP-2 receptor.
593
Cellular Physiology and Biochemistry
Cell Physiol Biochem 2014;34:590-602 DOI: 10.1159/000363025 Published online: August 11, 2014
© 2014 S. Karger AG, Basel www.karger.com/cpb
Xie et al.: GLP-2 Inhibits ERK and NF-kB Pathway to Suppress the Inflammation
Fig. 1. Effects of GLP-2 on LPS-induced expression of proteins and mRNA of iNOS and COX-2 in macrophages. Macrophages were pretreated with GLP-2 (10-9, 10-8, 10-7 and 10-6 M) 1 h prior to incubation of LPS (100 ng/mL) for 4 h. Proteins and mRNA of iNOS and COX-2 were determined by Western blotting and qRT-PCR. Panels A and B show mRNA of iNOS (A) and COX-2 (B). Panels C, D and E show the protein expression of iNOS (C, D) and COX-2 (C, E), with levels normalized to β-actin. The results are expressed as the means ± SD for each group from three independent experiments. # Significant compared to control alone, p
