- Email: [email protected]
Effects of hesperetin on the production of inflammatory mediators in IL-1β treated human synovial cells
Cellular Immunology 264 (2010) 1–3
Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Rapid Communication
Effects of hesperetin on the production of inflammatory mediators in IL-1b treated human synovial cells Eun Mi Choi *, Young Soon Lee Department of Food & Nutrition, Kyung Hee University, 1, Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 8 March 2010 Accepted 12 May 2010 Available online 19 May 2010 Keywords: Hesperetin SW982 Matrix metalloproteinases Cytokines Mitogen-activated protein kinases
a b s t r a c t This study was conducted to evaluate the efficacy of hesperetin in regulating interleukin-1b (IL-1b)-induced production of the matrix metalloproteinase (MMP)-3 and IL-6 in human synovial cell line, SW982. Treatment with hesperetin at 1 or 10 lM significantly (P < 0.05) inhibited IL-1b-induced MMP-3 and IL-6 production when measured by enzyme-linked immunosorbent assay (ELISA). The effects of hesperetin on the activation of mitogen-activated protein kinases (MAPKs) were also examined in SW982 cells by ELISA assay. IL-1b-induced JNK activation was inhibited by hesperetin. These results suggest that hesperetin reduces the production of MMP and IL-6 in SW982 synovial cells by inhibiting JNK. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease which is characterized by leukocyte recruitment and activation, cell proliferation, angiogenesis, and pannus formation ultimately resulting in joint destruction [1]. Abnormal proliferation of synovium and excessive secretion of pro-inflammatory cytokines by these cells account for many of the pathological changes seen in an RA joint [2]. The severity of the joint inflammation fluctuates over time, and the outcome of the uncontrolled disease is progressive joint destruction, deformity, and disability [1,2]. Synovial fibroblasts are important producers of inflammatory mediators such as cytokines and matrix metalloproteinases (MMPs) [3]. MMPs are responsible for the destruction of cartilage and bone. It is well known that pro-inflammatory cytokines such as IL-1b and IL-6 stimulate production of MMPs through the activation of cellular signaling pathways involving mitogen-activated protein kinases (MAPKs). Three major MAPK families that differ in their substrate specificity and responses to stress have been identified in vertebrates and have been implicated in RA: c-Jun N-terminal kinase (JNK), extracellular regulating kinase (ERK), and p38 kinase [4]. MAPKs phosphorylate selected intracellular proteins, including transcription factors, which subsequently regulate gene expression by transcriptional and posttranscriptional mechanisms [5]. It is well known that primary mammalian cells in culture have a finite replicative life span and eventually enter a state of
senescence in which they remain metabolically active but cease to proliferate, and routinely obtaining RA-derived synovial tissue samples is difficult. SW982 human synovial sarcoma cell line is characterized by expression of inflammatory cytokine and MMP genes [6]. SW982 cells express genes encoding IL-1b, IL-6, cyclooxygenase (COX)-2, and MMPs. The cells, however, failed to express COX-1 and MMP-9. The lack of MMP-9 in SW982 cells suggests that the cell line is actually derived from synovial fibroblasts. Cells in tightly packed confluent monolayers exhibit tissue-specific differentiated functions [6]. These suggest that SW982 cell line is a useful tool for the study of inflammatory cytokine and MMP. Hesperetin (30 ,5,7-trihydroxy-4-methoxyflavanone) is a member of the flavanone subclass of flavonoids and occurs in fruit sources including citrus species. Like most flavonoids, hesperetin exists in nature in its glycoside form, hesperidin. Dietary hesperidin is deglycosylated into hesperetin by intestinal bacteria before absorption. Hesperetin has been relatively unnoticed in comparison with hesperidin, and few in vitro studies have assessed it. In this study, we investigated the inhibitory effect of hesperetin on IL-1b-induced production of inflammatory mediators (MMP-3 and IL-6) as well as MAPK phosphorylation in RA inflammation using SW982 human synovial cells.
2. Materials and methods 2.1. Reagents
* Corresponding author. Fax: +82 2 968 0260. E-mail address: [email protected] (E.M. Choi). 0008-8749/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2010.05.006
Hesperetin was purchased from Wako Pure Chemicals, Industries, Ltd. (Japan). Hesperetin was dissolved in dimethylsulfoxide
E.M. Choi, Y.S. Lee / Cellular Immunology 264 (2010) 1–3
2.2. Cell culture The human synovial cell line SW982 was obtained from American Tissue Culture Collection (Manassas, VA). SW982 cells were routinely cultured in T-150 flasks (Corning) and grown in DMEM with 2 mM L-glutamine, 10% feral bovine serum (FBS) and 1% Pen-Strep (Invitrogen) at 37 °C, 5% CO2. 2.3. Determination of MMP-3 and IL-6 SW982 were plated in 24-well plates (105 cells per well) for 48 h before treatment. After washing with PBS (pH 7.4), cells were incubated at 37 °C with or without hesperetin and IL-1b (5 ng/ml) for 24 h in DMEM containing 10% (v/v) FBS in a 5% CO2 atmosphere. Culture supernatants were collected and stored at 80 °C. The productions of MMP-3 and IL-6 were determined in culture supernatant from the above experiments using commercially available ELISA kits essentially according to the instructions of the manufacturer using Human MMP-3 ELISA Kit (Ray BioÒ) and QuantikineÒ Human IL-6 immunoassay kit (R&D System). 2.4. Cell viability Cells were suspended in medium supplemented with 10% FBS, and cell suspension containing 5 103 cells was added to the individual wells of 48-well microplates. The plates were incubated at 37 °C in a CO2 incubator for 24 h. After discarding the culture medium and washing the cells with phosphate-buffered saline (PBS), serum-free medium containing 0.3% bovine serum albumin (BSA) and agents at appropriate concentrations was added to the cell culture and incubated at 37 °C in a humidified atmosphere of 5% CO2 for 48 h. Surviving cells was counted by the 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) method. MTT 20 ll in 7.2 mM phosphate buffer solution, pH 6.5 (5 mg/ml), was added to each well, and the plates were incubated for an additional 2 h. After the removal of solutions in the well, dimethyl sulfoxide was added to dissolve formazan products, and the plates were shaken for 5 min. The absorbance of each well was recorded on a microplate spectrophotometer at 570 nm.
3. Results and discussion MMP-3 (stromelysin) can degrade proteoglycan, type IV and IX collagens, denatured type I and II collagens, fibronectin, gelatin, and laminin and is considered to be especially important because, in addition to its direct enzyme activity, its activation is necessary for full activation of collagenases [7]. To evaluate the effect of hesperetin on IL-1b-induced production of MMP-3 protein, we treated SW982 synovial cells with IL-1b alone and with IL-1b + hesperetin for 24 h. The effect of hesperetin on IL-1b-induced MMP-3 production was determined by ELISA. As shown in Fig. 1A, SW982 synovial cells expressed a low level of MMP-3 without IL-1b treatment. Stimulation of SW982 cells with IL-1b (5 ng/ml) alone resulted in significant increase in MMP-3 production (P < 0.05), compared to the levels detected in untreated SW982 cells. Treatment of SW982 cells with hesperetin (1 and 10 lM) resulted in inhibition of IL-1b-induced-MMP-3 production to the basal level detected in untreated SW982 cells (P < 0.05), indicating that hesperetin has antiprotease potential. When the cell viability was checked using MTT-staining method, these concentrations of hesperetin had no effect on the viability of these cells (Fig. 2). In order to assess the effect of hesperetin on the production of cytokine related to the inflammatory reaction, the levels of IL-6 were measured by ELISA from the synovial cells. As shown in Fig. 1B, IL-1b increased significantly the levels of IL-6 compared with vehicle. However, pre-treatment with hesperetin (1 and 10 lM) resulted in a significant inhibition of IL-1b-induced IL-6 production, indicating that hesperetin specifically blocks IL-6 induction in IL-1b signaling. Tissue destruction in RA is associated with pro-inflammatory cytokines. IL-6 plays a pivotal role in immunoinflammatory reactions. In addition, high levels of IL-6 8
6
(DMSO) and then diluted with the medium (final DMSO concentration 60.05% (v/v)). All other reagents were from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise stated.
MMP-3 (pg/10 cells)
2
2.5. Determination of MAPK activation
2.6. Statistical analysis The results are expressed as mean ± SEM (n = 5). Statistical analysis was performed using one-way ANOVA (P < 0.05). The analysis was performed using SAS statistical software.
*
7
# 6 5
#
4 3
IL-1β (5 ng/ml) Hesperetin ( μM) 1.20
-
(B)
1.15
+ -
+ 1
+
10
* #
1.10
#
6
IL-6 (ng/10 cells)
The Ray BioÒ Cell-Based MAPK ELISA Kit (USA) was used for measuring the relative amount of JNK (Thr183/Tyr185) and p38 MAPK (Thr180/Tyr182) phosphorylation in cultured cells. In the Cell-Based MAPK ELISA kit, cells are seeded into a 96 well tissue culture plate. After washing with PBS (pH 7.4), cells were incubated at 37 °C with or without hesperetin for 24 h and IL-1b (5 ng/ml) for 30 min. The cells are fixed after various treatments. After blocking, anti-phospho-protein specific antibody or antipan-protein specific antibody (primary antibody) is pipetted into the wells and incubated. The wells are washed, and HRP-conjugated anti-mouse IgG (secondary antibody) is added to the wells. The wells are washed again, a TMB substrate solution is added to the wells and color develops in proportion to the amount of protein. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
(A)
1.05 1.00 0.95 0.90
IL-1β (5 ng/ml) Hesperetin (μM)
-
+ -
+
1
+
10
Fig. 1. Inhibitory effect of hesperetin on the production of MMP-3 and IL-6 by SW982 cells. The SW982 were cultured for 24 h with hesperetin and IL-1b (5 ng/ ml). Data are expressed as mean ± SEM (n = 5). P < 0.05; no treatment vs. control (IL-1b alone), #P < 0.05; control vs. hesperetin.
E.M. Choi, Y.S. Lee / Cellular Immunology 264 (2010) 1–3
*
120
Cell viability (%)
100 80 60 40 20 0
IL-1β (5 ng/ml) Hesperetin (μM)
-
-
1
-
+ -
10
+
+
1
10
Fig. 2. Effect of hesperetin on the viability of MC3T3-E1 cells. Data were expressed as a percentage of control cells without IL-1b. Asterisk, significantly different from each other (P < 0.05).
play a vital role in the development of many autoimmune diseases. Thus, inhibition of cytokine production forms the basis of some currently used RA drugs. IL-1b induces and/or enhances the production of IL-6 as well as MMP-3 in many mesenchymal cell types such as, synovial cells and macrophages [8]. Since it has been re-
*
Phospho-JNK/JNK
1.75 1.50 1.25 1.00 0.75
Acknowledgment
0.25
This work was supported by a Grant from the Kyung Hee University in 2010 (KHU-20100111).
0.00
IL-1 β
-
Phospho-p38/p38
0.25
+ -
+ +
*
0.20 0.15 0.10 0.05 0.00
IL-1β Hesperetin
ported that nuclear factor kappa B (NF-jB) plays an important role in the transcriptional activation of these pro-inflammatory mediators [9], suppression by hesperetin of MMP and IL-6 production is likely to be due to the direct modulation of NF-jB binding to the promoter regions of the genes coding for these proteins. Thus, further studies are needed to clarify the exact mode of action of hesperetin on the production of MMP and cytokine in human synovial fibroblasts. To determine whether the effects of hesperetin are attributable to the inhibition of MAPK activation, we examined the effects of hesperetin on MAPK in IL-1b-stimulated synovial fibroblasts. SW982 cells demonstrated a detectable basal level of phosphorylation of MAPK proteins, and SW982 cells stimulated with IL-b induced remarkably higher levels of phosphorylation of JNK and p38 in comparison to the cells which were not stimulated with IL-1b (Fig. 3). IL-1b stimulation induced JNK and p38 phosphorylation, demonstrating activation of the kinase. Treatment with hesperetin (10 lM) reduced IL-1b-induced phosphorylation of JNK, but not p38. The modulation of the activity of JNK by IL-1b suggests a more expression of genes that have AP-1 binding sites in their promoters because JNK binds the c-Jun. Thus JNK may be the most relevant kinase that is induced and activated by pro-inflammatory cytokines in synoviocytes as this has the potential for enhancing the production of MMPs and other mediators of cartilage degradation in an arthritic joint. This is supported by the results of a recent study showing that the inhibition of JNK correlated with less joint damage and remodeling in an animal model of arthritis [10]. Therefore, inhibition of the modulation of activation of JNK by hesperetin may be important for inhibiting the IL-1b-induced catabolic events in synoviocytes. In conclusion, treatment of SW982 cells with hesperetin inhibited significantly IL-1b-induced phosphorylation of JNK and induction of MMP-3 and IL-6, demonstrating that hesperetin can be used as an anti-inflammatory and immunomodulatory agent in RA patients.
#
0.50
Hesperetin
3
-
+ -
+ +
Fig. 3. ELISA quantification of phosphorylated JNK and p38 in IL-1b-stimulated SW982 pretreated with hesperetin. Cells were incubated with or without 10 lM hesperetin for 24 h and with IL-1b (5 ng/ml) for 30 min. The control represents cells treated by IL-1b alone. Cells were lysed and JNK and phospho-JNK, p38 and phospho-p38 were quantified with MAPK ELISA kits. Data are expressed as mean ± SEM (n = 5). P < 0.05; no treatment vs. control (IL-1b alone), #P < 0.05; control vs. hesperetin.
References [1] M. Feldmann, F.M. Brennan, R.N. Maini, Role of cytokines in rheumatoid arthritis, Annu. Rev. Immunol. 14 (1996) 397–440. [2] A.E. Koch, S.L. Kunkel, L.A. Harlow, B. Johnson, H.L. Evanoff, G.K. Haines, Enhanced production of monocyte chemoattractant protein-1 in rheumatoid arthritis, J. Clin. Invest. 90 (1992) 772–779. [3] T. Pap, U. Muller-Ladner, R.E. Gay, S. Gay, Fibroblast biology. Role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis, Arthritis Res. 2 (2000) 361–367. [4] M.J. Robinson, M.H. Cobb, Mitogen-activated protein kinase pathways, Curr. Opin. Cell Biol. 9 (1997) 180–185. [5] R. Seger, E.G. Krebs, The MAPK signaling cascade, FASEB J. 9 (1995) 726– 735. [6] T. Yamazaki, T. Yokoyama, H. Akatsu, T. Tukiyama, T. Tokiwa, Phenotypic characterization of a human synovial sarcoma cell line, SW982, and its response to dexamethasone, In Vitro Cell Dev. Biol. Anim. 39 (2003) 337– 339. [7] Y. Yamanishi, G.S. Firestein, Pathogenesis of rheumatoid arthritis: the role of synoviocytes, Rheum. Dis. Clin. North Am. 27 (2001) 355–371. [8] A. Ito, T. Nose, S. Takahashi, Y. Mori, Cyclooxygenase inhibitors augment the production of promatrix metalloproteinase 9 (progelatinase B) in rabbit articular chondrocytes, FEBS Lett. 360 (1995) 75–79. [9] D.M. Jue, K.I. Jeon, J.Y. Jeong, Nuclear factor kappa B (NF-jB) pathway as a therapeutic target in rheumatoid arthritis, J. Korean Med. Sci. 14 (1999) 231– 238. [10] Z. Han, D.L. Boyle, L. Chang, B. Bennett, M. Karin, L. Yang, A.M. Manning, G.S. Firestein, C-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis, J. Clin. Invest. 108 (2001) 73– 81.
Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Rapid Communication
Effects of hesperetin on the production of inflammatory mediators in IL-1b treated human synovial cells Eun Mi Choi *, Young Soon Lee Department of Food & Nutrition, Kyung Hee University, 1, Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 8 March 2010 Accepted 12 May 2010 Available online 19 May 2010 Keywords: Hesperetin SW982 Matrix metalloproteinases Cytokines Mitogen-activated protein kinases
a b s t r a c t This study was conducted to evaluate the efficacy of hesperetin in regulating interleukin-1b (IL-1b)-induced production of the matrix metalloproteinase (MMP)-3 and IL-6 in human synovial cell line, SW982. Treatment with hesperetin at 1 or 10 lM significantly (P < 0.05) inhibited IL-1b-induced MMP-3 and IL-6 production when measured by enzyme-linked immunosorbent assay (ELISA). The effects of hesperetin on the activation of mitogen-activated protein kinases (MAPKs) were also examined in SW982 cells by ELISA assay. IL-1b-induced JNK activation was inhibited by hesperetin. These results suggest that hesperetin reduces the production of MMP and IL-6 in SW982 synovial cells by inhibiting JNK. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease which is characterized by leukocyte recruitment and activation, cell proliferation, angiogenesis, and pannus formation ultimately resulting in joint destruction [1]. Abnormal proliferation of synovium and excessive secretion of pro-inflammatory cytokines by these cells account for many of the pathological changes seen in an RA joint [2]. The severity of the joint inflammation fluctuates over time, and the outcome of the uncontrolled disease is progressive joint destruction, deformity, and disability [1,2]. Synovial fibroblasts are important producers of inflammatory mediators such as cytokines and matrix metalloproteinases (MMPs) [3]. MMPs are responsible for the destruction of cartilage and bone. It is well known that pro-inflammatory cytokines such as IL-1b and IL-6 stimulate production of MMPs through the activation of cellular signaling pathways involving mitogen-activated protein kinases (MAPKs). Three major MAPK families that differ in their substrate specificity and responses to stress have been identified in vertebrates and have been implicated in RA: c-Jun N-terminal kinase (JNK), extracellular regulating kinase (ERK), and p38 kinase [4]. MAPKs phosphorylate selected intracellular proteins, including transcription factors, which subsequently regulate gene expression by transcriptional and posttranscriptional mechanisms [5]. It is well known that primary mammalian cells in culture have a finite replicative life span and eventually enter a state of
senescence in which they remain metabolically active but cease to proliferate, and routinely obtaining RA-derived synovial tissue samples is difficult. SW982 human synovial sarcoma cell line is characterized by expression of inflammatory cytokine and MMP genes [6]. SW982 cells express genes encoding IL-1b, IL-6, cyclooxygenase (COX)-2, and MMPs. The cells, however, failed to express COX-1 and MMP-9. The lack of MMP-9 in SW982 cells suggests that the cell line is actually derived from synovial fibroblasts. Cells in tightly packed confluent monolayers exhibit tissue-specific differentiated functions [6]. These suggest that SW982 cell line is a useful tool for the study of inflammatory cytokine and MMP. Hesperetin (30 ,5,7-trihydroxy-4-methoxyflavanone) is a member of the flavanone subclass of flavonoids and occurs in fruit sources including citrus species. Like most flavonoids, hesperetin exists in nature in its glycoside form, hesperidin. Dietary hesperidin is deglycosylated into hesperetin by intestinal bacteria before absorption. Hesperetin has been relatively unnoticed in comparison with hesperidin, and few in vitro studies have assessed it. In this study, we investigated the inhibitory effect of hesperetin on IL-1b-induced production of inflammatory mediators (MMP-3 and IL-6) as well as MAPK phosphorylation in RA inflammation using SW982 human synovial cells.
2. Materials and methods 2.1. Reagents
* Corresponding author. Fax: +82 2 968 0260. E-mail address: [email protected] (E.M. Choi). 0008-8749/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2010.05.006
Hesperetin was purchased from Wako Pure Chemicals, Industries, Ltd. (Japan). Hesperetin was dissolved in dimethylsulfoxide
E.M. Choi, Y.S. Lee / Cellular Immunology 264 (2010) 1–3
2.2. Cell culture The human synovial cell line SW982 was obtained from American Tissue Culture Collection (Manassas, VA). SW982 cells were routinely cultured in T-150 flasks (Corning) and grown in DMEM with 2 mM L-glutamine, 10% feral bovine serum (FBS) and 1% Pen-Strep (Invitrogen) at 37 °C, 5% CO2. 2.3. Determination of MMP-3 and IL-6 SW982 were plated in 24-well plates (105 cells per well) for 48 h before treatment. After washing with PBS (pH 7.4), cells were incubated at 37 °C with or without hesperetin and IL-1b (5 ng/ml) for 24 h in DMEM containing 10% (v/v) FBS in a 5% CO2 atmosphere. Culture supernatants were collected and stored at 80 °C. The productions of MMP-3 and IL-6 were determined in culture supernatant from the above experiments using commercially available ELISA kits essentially according to the instructions of the manufacturer using Human MMP-3 ELISA Kit (Ray BioÒ) and QuantikineÒ Human IL-6 immunoassay kit (R&D System). 2.4. Cell viability Cells were suspended in medium supplemented with 10% FBS, and cell suspension containing 5 103 cells was added to the individual wells of 48-well microplates. The plates were incubated at 37 °C in a CO2 incubator for 24 h. After discarding the culture medium and washing the cells with phosphate-buffered saline (PBS), serum-free medium containing 0.3% bovine serum albumin (BSA) and agents at appropriate concentrations was added to the cell culture and incubated at 37 °C in a humidified atmosphere of 5% CO2 for 48 h. Surviving cells was counted by the 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) method. MTT 20 ll in 7.2 mM phosphate buffer solution, pH 6.5 (5 mg/ml), was added to each well, and the plates were incubated for an additional 2 h. After the removal of solutions in the well, dimethyl sulfoxide was added to dissolve formazan products, and the plates were shaken for 5 min. The absorbance of each well was recorded on a microplate spectrophotometer at 570 nm.
3. Results and discussion MMP-3 (stromelysin) can degrade proteoglycan, type IV and IX collagens, denatured type I and II collagens, fibronectin, gelatin, and laminin and is considered to be especially important because, in addition to its direct enzyme activity, its activation is necessary for full activation of collagenases [7]. To evaluate the effect of hesperetin on IL-1b-induced production of MMP-3 protein, we treated SW982 synovial cells with IL-1b alone and with IL-1b + hesperetin for 24 h. The effect of hesperetin on IL-1b-induced MMP-3 production was determined by ELISA. As shown in Fig. 1A, SW982 synovial cells expressed a low level of MMP-3 without IL-1b treatment. Stimulation of SW982 cells with IL-1b (5 ng/ml) alone resulted in significant increase in MMP-3 production (P < 0.05), compared to the levels detected in untreated SW982 cells. Treatment of SW982 cells with hesperetin (1 and 10 lM) resulted in inhibition of IL-1b-induced-MMP-3 production to the basal level detected in untreated SW982 cells (P < 0.05), indicating that hesperetin has antiprotease potential. When the cell viability was checked using MTT-staining method, these concentrations of hesperetin had no effect on the viability of these cells (Fig. 2). In order to assess the effect of hesperetin on the production of cytokine related to the inflammatory reaction, the levels of IL-6 were measured by ELISA from the synovial cells. As shown in Fig. 1B, IL-1b increased significantly the levels of IL-6 compared with vehicle. However, pre-treatment with hesperetin (1 and 10 lM) resulted in a significant inhibition of IL-1b-induced IL-6 production, indicating that hesperetin specifically blocks IL-6 induction in IL-1b signaling. Tissue destruction in RA is associated with pro-inflammatory cytokines. IL-6 plays a pivotal role in immunoinflammatory reactions. In addition, high levels of IL-6 8
6
(DMSO) and then diluted with the medium (final DMSO concentration 60.05% (v/v)). All other reagents were from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise stated.
MMP-3 (pg/10 cells)
2
2.5. Determination of MAPK activation
2.6. Statistical analysis The results are expressed as mean ± SEM (n = 5). Statistical analysis was performed using one-way ANOVA (P < 0.05). The analysis was performed using SAS statistical software.
*
7
# 6 5
#
4 3
IL-1β (5 ng/ml) Hesperetin ( μM) 1.20
-
(B)
1.15
+ -
+ 1
+
10
* #
1.10
#
6
IL-6 (ng/10 cells)
The Ray BioÒ Cell-Based MAPK ELISA Kit (USA) was used for measuring the relative amount of JNK (Thr183/Tyr185) and p38 MAPK (Thr180/Tyr182) phosphorylation in cultured cells. In the Cell-Based MAPK ELISA kit, cells are seeded into a 96 well tissue culture plate. After washing with PBS (pH 7.4), cells were incubated at 37 °C with or without hesperetin for 24 h and IL-1b (5 ng/ml) for 30 min. The cells are fixed after various treatments. After blocking, anti-phospho-protein specific antibody or antipan-protein specific antibody (primary antibody) is pipetted into the wells and incubated. The wells are washed, and HRP-conjugated anti-mouse IgG (secondary antibody) is added to the wells. The wells are washed again, a TMB substrate solution is added to the wells and color develops in proportion to the amount of protein. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
(A)
1.05 1.00 0.95 0.90
IL-1β (5 ng/ml) Hesperetin (μM)
-
+ -
+
1
+
10
Fig. 1. Inhibitory effect of hesperetin on the production of MMP-3 and IL-6 by SW982 cells. The SW982 were cultured for 24 h with hesperetin and IL-1b (5 ng/ ml). Data are expressed as mean ± SEM (n = 5). P < 0.05; no treatment vs. control (IL-1b alone), #P < 0.05; control vs. hesperetin.
E.M. Choi, Y.S. Lee / Cellular Immunology 264 (2010) 1–3
*
120
Cell viability (%)
100 80 60 40 20 0
IL-1β (5 ng/ml) Hesperetin (μM)
-
-
1
-
+ -
10
+
+
1
10
Fig. 2. Effect of hesperetin on the viability of MC3T3-E1 cells. Data were expressed as a percentage of control cells without IL-1b. Asterisk, significantly different from each other (P < 0.05).
play a vital role in the development of many autoimmune diseases. Thus, inhibition of cytokine production forms the basis of some currently used RA drugs. IL-1b induces and/or enhances the production of IL-6 as well as MMP-3 in many mesenchymal cell types such as, synovial cells and macrophages [8]. Since it has been re-
*
Phospho-JNK/JNK
1.75 1.50 1.25 1.00 0.75
Acknowledgment
0.25
This work was supported by a Grant from the Kyung Hee University in 2010 (KHU-20100111).
0.00
IL-1 β
-
Phospho-p38/p38
0.25
+ -
+ +
*
0.20 0.15 0.10 0.05 0.00
IL-1β Hesperetin
ported that nuclear factor kappa B (NF-jB) plays an important role in the transcriptional activation of these pro-inflammatory mediators [9], suppression by hesperetin of MMP and IL-6 production is likely to be due to the direct modulation of NF-jB binding to the promoter regions of the genes coding for these proteins. Thus, further studies are needed to clarify the exact mode of action of hesperetin on the production of MMP and cytokine in human synovial fibroblasts. To determine whether the effects of hesperetin are attributable to the inhibition of MAPK activation, we examined the effects of hesperetin on MAPK in IL-1b-stimulated synovial fibroblasts. SW982 cells demonstrated a detectable basal level of phosphorylation of MAPK proteins, and SW982 cells stimulated with IL-b induced remarkably higher levels of phosphorylation of JNK and p38 in comparison to the cells which were not stimulated with IL-1b (Fig. 3). IL-1b stimulation induced JNK and p38 phosphorylation, demonstrating activation of the kinase. Treatment with hesperetin (10 lM) reduced IL-1b-induced phosphorylation of JNK, but not p38. The modulation of the activity of JNK by IL-1b suggests a more expression of genes that have AP-1 binding sites in their promoters because JNK binds the c-Jun. Thus JNK may be the most relevant kinase that is induced and activated by pro-inflammatory cytokines in synoviocytes as this has the potential for enhancing the production of MMPs and other mediators of cartilage degradation in an arthritic joint. This is supported by the results of a recent study showing that the inhibition of JNK correlated with less joint damage and remodeling in an animal model of arthritis [10]. Therefore, inhibition of the modulation of activation of JNK by hesperetin may be important for inhibiting the IL-1b-induced catabolic events in synoviocytes. In conclusion, treatment of SW982 cells with hesperetin inhibited significantly IL-1b-induced phosphorylation of JNK and induction of MMP-3 and IL-6, demonstrating that hesperetin can be used as an anti-inflammatory and immunomodulatory agent in RA patients.
#
0.50
Hesperetin
3
-
+ -
+ +
Fig. 3. ELISA quantification of phosphorylated JNK and p38 in IL-1b-stimulated SW982 pretreated with hesperetin. Cells were incubated with or without 10 lM hesperetin for 24 h and with IL-1b (5 ng/ml) for 30 min. The control represents cells treated by IL-1b alone. Cells were lysed and JNK and phospho-JNK, p38 and phospho-p38 were quantified with MAPK ELISA kits. Data are expressed as mean ± SEM (n = 5). P < 0.05; no treatment vs. control (IL-1b alone), #P < 0.05; control vs. hesperetin.
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