marine drugs Article
The Oligo Fucoidan Inhibits Platelet-Derived Growth Factor-Stimulated Proliferation of Airway Smooth Muscle Cells Chao-Huei Yang 1 , Chiung-Fang Tsao 2 , Wang-Sheng Ko 1,3, * and Ya-Ling Chiou 3,4, * Received: 12 October 2015; Accepted: 4 January 2016; Published: 9 January 2016 Academic Editor: Paola Laurienzo 1 2 3 4
*
Department of Internal Medicine, Kuang-Tien General Hospital, No. 117, Shatian Road Shalu District, Taichung City 433, Taiwan; [email protected] Department of Biotechnology, Hungkuang University, 34 Chung-Chie Rd, Sha Lu, Taichung 443, Taiwan; [email protected] Institute of BioMedical Nutrition, Hungkuang University, 34 Chung-Chie Rd, Sha Lu, Taichung 443, Taiwan Department of Nursing, Hungkuang University, 34 Chung-Chie Rd, Sha Lu, Taichung 443, Taiwan Correspondence: [email protected] (W.-S.K.); [email protected] (Y.-L.C.); Tel.: +886-4-2631-8652 (ext. 3125) (W.-S.K. & Y.-L.C.); Fax: +886-4-2633-8212 (W.-S.K. & Y.-L.C.)
Abstract: In the pathogenesis of asthma, the proliferation of airway smooth muscle cells (ASMCs) is a key factor in airway remodeling and causes airway narrowing. In addition, ASMCs are also the effector cells of airway inflammation. Fucoidan extracted from marine brown algae polysaccharides has antiviral, antioxidant, antimicrobial, anticlotting, and anticancer properties; however, its effectiveness for asthma has not been elucidated thus far. Platelet-derived growth factor (PDGF)-treated primary ASMCs were cultured with or without oligo-fucoidan (100, 500, or 1000 µg/mL) to evaluate its effects on cell proliferation, cell cycle, apoptosis, and Akt, ERK1/2 signaling pathway. We found that PDGF (40 ng/mL) increased the proliferation of ASMCs by 2.5-fold after 48 h (p < 0.05). Oligo-fucoidan reduced the proliferation of PDGF-stimulated ASMCs by 75%–99% after 48 h (p < 0.05) and induced G1 /G0 cell cycle arrest, but did not induce apoptosis. Further, oligo-fucoidan supplementation reduced PDGF-stimulated extracellular signal-regulated kinase (ERK1/2), Akt, and nuclear factor (NF)-κB phosphorylation. Taken together, oligo-fucoidan supplementation might reduce proliferation of PDGF-treated ASMCs through the suppression of ERK1/2 and Akt phosphorylation and NF-κB activation. The results provide basis for future animal experiments and human trials. Keywords: asthma; airway smooth muscle cells; oligo-fucoidan
1. Introduction In 1974, the morbidity rate of childhood asthma in Taiwan was approximately 3%, which increased to approximately 20.74% by 2009, indicating a 7-fold increase in 35 years [1] This shows that asthma has become a common and serious issue Taiwan. In the pathogenesis of asthma, alteration in the structure of the airways is commonly observed resulting in airway smooth muscle cell hypertrophy, hyperplasia, and airway wall thickening. Recent studies have demonstrated that airway smooth muscle cells (ASMCs) are not just involved in airway narrowing. Stimulated ASMCs release interleukin (IL)-1, IL-5, IL-6, IL-8, IL-11, granulocyte monocyte-colony stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), and monocyte chemoattractant protein (MCP)-1, 2, 3. The stimulated ASMCs also express cell adhesion molecules (CAMs), intercellular CAM (ICAM), and vascular CAM (VCAM) to activate mast cells, eosinophils, lymphocytes, and neutrophil causing airway inflammation [2]; thus, the majority of asthma patients require permanent therapy with inhaled corticosteroids (ICS) or ICS + Mar. Drugs 2016, 14, 15; doi:10.3390/md14010015
www.mdpi.com/journal/marinedrugs
Mar. Drugs 2016, 14, 15
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long-acting beta2-agonists (LABA) to ameliorate airway inflammation [3]. Therefore, reducing the proliferation of ASMCs is one approach of reducing airway inflammation. In asthma pathogenesis, the injured epithelial tissue releases several factors, including endothelin-1, epidermal growth factor, insulin-like growth factor, and platelet-derived growth factor (PDGF) to induce the proliferation of ASMCs [4–6]. PDGF is a major factor in proliferation of ASMCs [7–9]. The PDGF family consists of five different isoforms, including PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD [10]. Both in vivo and in vitro studies showed that PDGF-BB induced the proliferation and migration of human ASMCs [11–13]. Therefore, the increased level of PDGF-BB in the airway of asthmatic patients was associated with changes in airway structure and function. PDGF can induce proliferation of ASMCs in a dose-dependent manner by activating various signaling pathways, including the mitogen activated protein kinase (MAPK), phosphoinositide-3-kinase (PI3K), and nuclear factor (NF)-κB pathways [14–16]. The proliferation of ASMCs is believed to be important in causing airway hyper-responsiveness (AHR), a prominent feature of airway remodeling [9]. Fucoidan is a seaweed polysaccharide derived from brown seaweed extract. It is structurally similar to heparin and consists of an alpha-1,3-backbone or a repeat units of disaccharides containing alpha-1,3-linked fucose and an alpha-1,4-linked fucose, with branching at the C2 position [17,18]. The fucoidan polysaccharide is reported to have antiviral, antioxidant, antimicrobial, anticoagulant, anticancer/antitumor, antiproliferative, and anti-inflammatory properties [18,19]. The use of fucoidan from various seaweed extracts for atopic allergic reactions was found to enhance the natural immune response, alter Th1/Th2 balance, inhibit immunoglobulin E (IgE) production, and suppress mast cell degranulation. Because fucoidan has these immunomodulatory effects, it has the potential to prevent allergic diseases [20,21]. However, the effects of oligo-fucoidan on allergic disease have not been elucidated thus far. The proliferation of ASMCs is a key factor in causing AHR. In ASMCs, the regulation of cell proliferation signals closely influences pathological processes. Therefore, we aimed to investigate the effects of oligo-fucoidan on the proliferation of PDGF-treated ASMCs. We used PDGF to induce the proliferation of ASMCs and found that treatment with oligo-fucoidan (100, 500, or 1000 µg/mL) inhibited the proliferation of PDGF-treated ASMCs. Oligo-fucoidan inhibited ERK1/2, Akt, and NF-κB phosphorylation in PDGF-treated ASMCs. Therefore, oligo-fucoidan may decrease the proliferation of PDGF-treated ASMCs by suppressing the ERK1/2, Akt, and NF-κB pathways. 2. Results 2.1. Oligo-Fucoidan Inhibited PDGF-Stimulated Proliferation of ASMCs To evaluate the effect of oligo-fucoidan on PDGF-stimulated proliferation of ASMCs, growth-arrested ASMCs were treated with 40-ng/mL rhPDGF-BB or oligo fucoidan (100, 500, or 1000 µg/mL). After incubation for 48 h, PDGF at 40-ng/mL stimulated marked proliferation of ASMCs. No significant differences in the proliferation rate of oligo-fucoidan alone treated ASMCs compared to the control group. Treatment with oligo-fucoidan alone did not affect the growth of cells cultured in 0.2% FBS-containing medium. (Figure 1a). Growth-arrested cells were treated with 40-ng/mL PDGF and oligo-fucoidan (100, 500, or 1000 µg/mL) for 24 h, 48 h, or 72 h. The proliferation fold of ASMCs decreased in 40 ng/mL PDGF and oligo-fucoidan groups significantly (Figure 1b). These results indicated that oligo-fucoidan inhibited the proliferation of PDGF-stimulated ASMCs. To evaluate the effect of oligo-fucoidan cpmpare with budesonide and fenoterol on PDGF-stimulated proliferation of ASMCs, Growth-arrested cells were treated with 40-ng/mL PDGF, 10 nM budesonide, 10 nM fenoterol and oligo-fucoidan (100, 500, or 1000 µg/mL) for 24 h and 48 h. The proliferation fold of PDGF-stumlated ASMCs did not decrease in budesonide and combination groups (10 nM budesonide + 10 nM fenoterol), except fenoterol group (p < 0.05) (Figure 1c). However, the combinatic effect of oligo-fucoidan (500 or 1000 µg/mL) reduced the proliferation fold of PDGF-stumlated ASMCs in budesonide, fenoterol and combination groups significantly (p < 0.05). These results indicated that
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0.05).These results indicated that oligo‐fucoidan significantly improve the effects of budesonide and oligo-fucoidan significantly improve the effects of budesonide and fenoterol in the proliferation of fenoterol in the proliferation of PDGF‐stimulated ASMCs. PDGF-stimulated ASMCs.
(a)
(b) Figure 1. Cont. Figure 1. Cont.
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(c) Figure 1. The oligo Fucoidan inhibited platelet‐derived growth factor (PDGF)‐stimulated Figure 1. The oligo Fucoidan inhibited platelet-derived growth factor (PDGF)-stimulated proliferation proliferation of airway smooth muscle cells (ASMCs). ASMCs were serum deprivation (0.2% of airway smooth muscle cells (ASMCs). ASMCs were serum deprivation (0.2% FBS-containing FBS‐containing medium) for 48 h, growth‐arrested ASMCs were treated with 40‐ng/mL rhPDGF‐BB medium) for 48 h, growth-arrested ASMCs were treated with 40-ng/mL rhPDGF-BB or oligo fucoidan or oligo fucoidan (100, 500, or 1000 μg/mL). After incubation for 48 h, the rate of proliferation of cells (100, 500, or 1000 µg/mL). After incubation for 48 h, the rate of proliferation of cells was assayed was assayed by XTT. (a) Growth‐arrested cells were treated with 40‐ng/mL rhPDGF‐BB and oligo by XTT. (a) Growth-arrested cells were treated with 40-ng/mL rhPDGF-BB and oligo fucoidan (100, fucoidan (100, 500, or 1000 μg/mL) for 24 h, 48 h, or 72 h, the rate of proliferation of cells was assayed 500, or 1000 µg/mL) for 24 h, 48 h, or 72 h, the rate of proliferation of cells was assayed by XTT; by XTT; (b) Growth‐arrested cells were treated with 40‐ng/mL rhPDGF‐BB, 40‐ng/mL rhPDGF‐BB + (b) Growth-arrested cells were treated with 40-ng/mL rhPDGF-BB, 40-ng/mL rhPDGF-BB + 10 nM 10 nM budesonide, rhPDGF‐BB + 10 nM 40‐ng/mL rhPDGF‐BB budesonide, 40-ng/mL40‐ng/mL rhPDGF-BB + 10 nM fenoterol and fenoterol 40-ng/mLand rhPDGF-BB + combination +combination (10 nM + 10 nM or fenoterol) withot or with different oligo fucoidan (100, (10 nM budesonide + 10budesonide nM fenoterol) withot with different oligo fucoidan (100, 500, or 1000 µg/mL) μg/mL) concentration for 48 h, the rate of proliferation of cells was assayed XTT.* 500, or 1000 concentration for 48 h, the rate of proliferation of cells was assayed by XTT.* compare withby control compare with control group (p
The Oligo Fucoidan Inhibits Platelet-Derived Growth Factor-Stimulated Proliferation of Airway Smooth Muscle Cells Chao-Huei Yang 1 , Chiung-Fang Tsao 2 , Wang-Sheng Ko 1,3, * and Ya-Ling Chiou 3,4, * Received: 12 October 2015; Accepted: 4 January 2016; Published: 9 January 2016 Academic Editor: Paola Laurienzo 1 2 3 4
*
Department of Internal Medicine, Kuang-Tien General Hospital, No. 117, Shatian Road Shalu District, Taichung City 433, Taiwan; [email protected] Department of Biotechnology, Hungkuang University, 34 Chung-Chie Rd, Sha Lu, Taichung 443, Taiwan; [email protected] Institute of BioMedical Nutrition, Hungkuang University, 34 Chung-Chie Rd, Sha Lu, Taichung 443, Taiwan Department of Nursing, Hungkuang University, 34 Chung-Chie Rd, Sha Lu, Taichung 443, Taiwan Correspondence: [email protected] (W.-S.K.); [email protected] (Y.-L.C.); Tel.: +886-4-2631-8652 (ext. 3125) (W.-S.K. & Y.-L.C.); Fax: +886-4-2633-8212 (W.-S.K. & Y.-L.C.)
Abstract: In the pathogenesis of asthma, the proliferation of airway smooth muscle cells (ASMCs) is a key factor in airway remodeling and causes airway narrowing. In addition, ASMCs are also the effector cells of airway inflammation. Fucoidan extracted from marine brown algae polysaccharides has antiviral, antioxidant, antimicrobial, anticlotting, and anticancer properties; however, its effectiveness for asthma has not been elucidated thus far. Platelet-derived growth factor (PDGF)-treated primary ASMCs were cultured with or without oligo-fucoidan (100, 500, or 1000 µg/mL) to evaluate its effects on cell proliferation, cell cycle, apoptosis, and Akt, ERK1/2 signaling pathway. We found that PDGF (40 ng/mL) increased the proliferation of ASMCs by 2.5-fold after 48 h (p < 0.05). Oligo-fucoidan reduced the proliferation of PDGF-stimulated ASMCs by 75%–99% after 48 h (p < 0.05) and induced G1 /G0 cell cycle arrest, but did not induce apoptosis. Further, oligo-fucoidan supplementation reduced PDGF-stimulated extracellular signal-regulated kinase (ERK1/2), Akt, and nuclear factor (NF)-κB phosphorylation. Taken together, oligo-fucoidan supplementation might reduce proliferation of PDGF-treated ASMCs through the suppression of ERK1/2 and Akt phosphorylation and NF-κB activation. The results provide basis for future animal experiments and human trials. Keywords: asthma; airway smooth muscle cells; oligo-fucoidan
1. Introduction In 1974, the morbidity rate of childhood asthma in Taiwan was approximately 3%, which increased to approximately 20.74% by 2009, indicating a 7-fold increase in 35 years [1] This shows that asthma has become a common and serious issue Taiwan. In the pathogenesis of asthma, alteration in the structure of the airways is commonly observed resulting in airway smooth muscle cell hypertrophy, hyperplasia, and airway wall thickening. Recent studies have demonstrated that airway smooth muscle cells (ASMCs) are not just involved in airway narrowing. Stimulated ASMCs release interleukin (IL)-1, IL-5, IL-6, IL-8, IL-11, granulocyte monocyte-colony stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), and monocyte chemoattractant protein (MCP)-1, 2, 3. The stimulated ASMCs also express cell adhesion molecules (CAMs), intercellular CAM (ICAM), and vascular CAM (VCAM) to activate mast cells, eosinophils, lymphocytes, and neutrophil causing airway inflammation [2]; thus, the majority of asthma patients require permanent therapy with inhaled corticosteroids (ICS) or ICS + Mar. Drugs 2016, 14, 15; doi:10.3390/md14010015
www.mdpi.com/journal/marinedrugs
Mar. Drugs 2016, 14, 15
2 of 12
long-acting beta2-agonists (LABA) to ameliorate airway inflammation [3]. Therefore, reducing the proliferation of ASMCs is one approach of reducing airway inflammation. In asthma pathogenesis, the injured epithelial tissue releases several factors, including endothelin-1, epidermal growth factor, insulin-like growth factor, and platelet-derived growth factor (PDGF) to induce the proliferation of ASMCs [4–6]. PDGF is a major factor in proliferation of ASMCs [7–9]. The PDGF family consists of five different isoforms, including PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD [10]. Both in vivo and in vitro studies showed that PDGF-BB induced the proliferation and migration of human ASMCs [11–13]. Therefore, the increased level of PDGF-BB in the airway of asthmatic patients was associated with changes in airway structure and function. PDGF can induce proliferation of ASMCs in a dose-dependent manner by activating various signaling pathways, including the mitogen activated protein kinase (MAPK), phosphoinositide-3-kinase (PI3K), and nuclear factor (NF)-κB pathways [14–16]. The proliferation of ASMCs is believed to be important in causing airway hyper-responsiveness (AHR), a prominent feature of airway remodeling [9]. Fucoidan is a seaweed polysaccharide derived from brown seaweed extract. It is structurally similar to heparin and consists of an alpha-1,3-backbone or a repeat units of disaccharides containing alpha-1,3-linked fucose and an alpha-1,4-linked fucose, with branching at the C2 position [17,18]. The fucoidan polysaccharide is reported to have antiviral, antioxidant, antimicrobial, anticoagulant, anticancer/antitumor, antiproliferative, and anti-inflammatory properties [18,19]. The use of fucoidan from various seaweed extracts for atopic allergic reactions was found to enhance the natural immune response, alter Th1/Th2 balance, inhibit immunoglobulin E (IgE) production, and suppress mast cell degranulation. Because fucoidan has these immunomodulatory effects, it has the potential to prevent allergic diseases [20,21]. However, the effects of oligo-fucoidan on allergic disease have not been elucidated thus far. The proliferation of ASMCs is a key factor in causing AHR. In ASMCs, the regulation of cell proliferation signals closely influences pathological processes. Therefore, we aimed to investigate the effects of oligo-fucoidan on the proliferation of PDGF-treated ASMCs. We used PDGF to induce the proliferation of ASMCs and found that treatment with oligo-fucoidan (100, 500, or 1000 µg/mL) inhibited the proliferation of PDGF-treated ASMCs. Oligo-fucoidan inhibited ERK1/2, Akt, and NF-κB phosphorylation in PDGF-treated ASMCs. Therefore, oligo-fucoidan may decrease the proliferation of PDGF-treated ASMCs by suppressing the ERK1/2, Akt, and NF-κB pathways. 2. Results 2.1. Oligo-Fucoidan Inhibited PDGF-Stimulated Proliferation of ASMCs To evaluate the effect of oligo-fucoidan on PDGF-stimulated proliferation of ASMCs, growth-arrested ASMCs were treated with 40-ng/mL rhPDGF-BB or oligo fucoidan (100, 500, or 1000 µg/mL). After incubation for 48 h, PDGF at 40-ng/mL stimulated marked proliferation of ASMCs. No significant differences in the proliferation rate of oligo-fucoidan alone treated ASMCs compared to the control group. Treatment with oligo-fucoidan alone did not affect the growth of cells cultured in 0.2% FBS-containing medium. (Figure 1a). Growth-arrested cells were treated with 40-ng/mL PDGF and oligo-fucoidan (100, 500, or 1000 µg/mL) for 24 h, 48 h, or 72 h. The proliferation fold of ASMCs decreased in 40 ng/mL PDGF and oligo-fucoidan groups significantly (Figure 1b). These results indicated that oligo-fucoidan inhibited the proliferation of PDGF-stimulated ASMCs. To evaluate the effect of oligo-fucoidan cpmpare with budesonide and fenoterol on PDGF-stimulated proliferation of ASMCs, Growth-arrested cells were treated with 40-ng/mL PDGF, 10 nM budesonide, 10 nM fenoterol and oligo-fucoidan (100, 500, or 1000 µg/mL) for 24 h and 48 h. The proliferation fold of PDGF-stumlated ASMCs did not decrease in budesonide and combination groups (10 nM budesonide + 10 nM fenoterol), except fenoterol group (p < 0.05) (Figure 1c). However, the combinatic effect of oligo-fucoidan (500 or 1000 µg/mL) reduced the proliferation fold of PDGF-stumlated ASMCs in budesonide, fenoterol and combination groups significantly (p < 0.05). These results indicated that
Mar. Drugs 2016, 14, 15 Mar. Drugs 2016, 14, x
3 of 12 3 of 12
0.05).These results indicated that oligo‐fucoidan significantly improve the effects of budesonide and oligo-fucoidan significantly improve the effects of budesonide and fenoterol in the proliferation of fenoterol in the proliferation of PDGF‐stimulated ASMCs. PDGF-stimulated ASMCs.
(a)
(b) Figure 1. Cont. Figure 1. Cont.
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(c) Figure 1. The oligo Fucoidan inhibited platelet‐derived growth factor (PDGF)‐stimulated Figure 1. The oligo Fucoidan inhibited platelet-derived growth factor (PDGF)-stimulated proliferation proliferation of airway smooth muscle cells (ASMCs). ASMCs were serum deprivation (0.2% of airway smooth muscle cells (ASMCs). ASMCs were serum deprivation (0.2% FBS-containing FBS‐containing medium) for 48 h, growth‐arrested ASMCs were treated with 40‐ng/mL rhPDGF‐BB medium) for 48 h, growth-arrested ASMCs were treated with 40-ng/mL rhPDGF-BB or oligo fucoidan or oligo fucoidan (100, 500, or 1000 μg/mL). After incubation for 48 h, the rate of proliferation of cells (100, 500, or 1000 µg/mL). After incubation for 48 h, the rate of proliferation of cells was assayed was assayed by XTT. (a) Growth‐arrested cells were treated with 40‐ng/mL rhPDGF‐BB and oligo by XTT. (a) Growth-arrested cells were treated with 40-ng/mL rhPDGF-BB and oligo fucoidan (100, fucoidan (100, 500, or 1000 μg/mL) for 24 h, 48 h, or 72 h, the rate of proliferation of cells was assayed 500, or 1000 µg/mL) for 24 h, 48 h, or 72 h, the rate of proliferation of cells was assayed by XTT; by XTT; (b) Growth‐arrested cells were treated with 40‐ng/mL rhPDGF‐BB, 40‐ng/mL rhPDGF‐BB + (b) Growth-arrested cells were treated with 40-ng/mL rhPDGF-BB, 40-ng/mL rhPDGF-BB + 10 nM 10 nM budesonide, rhPDGF‐BB + 10 nM 40‐ng/mL rhPDGF‐BB budesonide, 40-ng/mL40‐ng/mL rhPDGF-BB + 10 nM fenoterol and fenoterol 40-ng/mLand rhPDGF-BB + combination +combination (10 nM + 10 nM or fenoterol) withot or with different oligo fucoidan (100, (10 nM budesonide + 10budesonide nM fenoterol) withot with different oligo fucoidan (100, 500, or 1000 µg/mL) μg/mL) concentration for 48 h, the rate of proliferation of cells was assayed XTT.* 500, or 1000 concentration for 48 h, the rate of proliferation of cells was assayed by XTT.* compare withby control compare with control group (p
