Dietary factors play significant aetiological roles in the development of several diseases. Many epidemiological studies have shown an inverse relationship between the consumption of diets rich in polyphenols, especially flavonoids, and cardiovascular risk(Reference Tunstall-Pedoe, Kuulasmaa and Amouyel1). CVD in the Far East is significantly lower than that documented in the West, and atherosclerosis is the leading cause of morbidity and mortality in Western society(Reference Tunstall-Pedoe, Kuulasmaa and Amouyel1).
Recently, some independent laboratories have focused their attention on other dietary compounds, such as serotonin derivatives, which may exert significant antioxidant effects both in vitro and in vivo. N-(p-coumaroyl)serotonin (CS) and N-feruloylserotonin (FS), two serotonin conjugates, were identified as the major and unique phenolic constituents of defatted safflower seeds(Reference Koyama, Kuribayashi and Seki2, Reference Zhang, Nagatsu and Watanabe3). These are members of the indole hydroxycinnamic acid amides, known to perform defensive functions in plants when damaged or under pathogenic attack, with serotonin (5-HT), p-coumaric acid (p-ca) and ferulic acid (fa) representing the components of their structure (Fig. 1)(Reference Ly, Kang and Choi4). The hydroxycinnamic acid amides of serotonin, including CS and FS, which are synthesised by serotonin N-hydroxycinnamoyltransferase, are present in several wild-growing plants and their seeds, such as safflower (Carthamus tinctorius L.) used in herbal medicine in Eastern countries, and worldwide mainly for edible oil production(Reference Zhang, Nagatsu and Watanabe3, Reference Ly, Kang and Choi4), konnyaku (Amorphophallus konjac K. Koch) widely used as a traditional food in all over Japan(Reference Niwa, Murakami and Ohtake5), Japanese barnyard millet (Echinochloa utilis Ohwi & Yabuno), widely used in Japan especially in cold districts because of its resistance to low temperatures and for its highly nutritious grains that make it a good substitute for rice(Reference Watanabe6), Centaurea nigra L. and many species of the genus Centaurea, traditionally used for their antibacterial activity in the treatment of various aliments(Reference Kumarasamy, Middleton and Reid7–Reference Sarker, Savchenko and Whiting9), and maize bran and other grains used in a wide variety of ways, including home cooking to increase the fibre content of various foods(Reference Choi, Lee and Kim10). Furthermore, their presence was also detected in several vegetables(Reference Ly, Kang and Choi4) widely used all over the world.
Chemical structure of N-(p-coumaroyl)serotonin and N-feruloylserotonin.
Some of their activities have been already reported; they possess antibacterial and free radical-scavenging activities(Reference Niwa, Murakami and Ohtake5–Reference Kumarasamy, Middleton and Reid7, Reference Choi, Lee and Kim10), show antioxidative activity on plasma and liver lipid status(Reference Cho, Lee and Kim11), increase the proliferation of fibroblasts(Reference Kawashima, Hayashi and Takii12), inhibit the production of pro-inflammatory cytokines(Reference Kawashima, Hayashi and Takii12), inhibit LDL oxidation in apoE-deficient mice(Reference Koyama, Kuribayashi and Seki2) and show beneficial effects against cardiovascular risk in healthy human volunteers(Reference Koyama, Suzuki and Furukawa13). Despite this, there are no reports describing a cell-based mechanism of these serotonin derivatives against endothelial damage; their protective effect in this system at a cellular level is still unknown.
Short-term high glucose exposure, even in terms of hours, could represent a dangerous stressor in the early stages of atherosclerogenesis, suggesting that its dangerous effect could also affect healthy individuals(Reference Piga, Naito and Kokura14). Monocyte–endothelial cell interactions play a pivotal role in the initiation and progression of an inflammatory process induced by several stressors, including hyperglycaemia, making human aortic endothelial cells (HAEC) and the human monocyte U-937 cell line suitable for this kind of approach.
Therefore, in order to look into the still unknown cellular mechanism and effect of serotonin derivatives, the present study followed a cell-based approach to underline the potential properties of CS and FS, focusing on their action against the increase of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1).
Materials and methods
Cells
HAEC (Clonetics, San Diego, CA, USA) were routinely maintained at 37°C in a humidified atmosphere of 95 % air and 5 % CO2 in endothelial cell growth medium (EBM-2; Clonetics) containing 5·5 mm-d-(+)-glucose, and supplemented with endothelial growth medium 2 (EGM-2) kit (Clonetics). Therefore, HAEC growing in their EBM-2 medium containing 5·5 mm-d-(+)-glucose required for their survival were used as control cells and considered as untreated. For all experiments, cells were used up to the sixth passage.
Human monocyte line U-937 (American Type Culture Collection, Rockville, MD, USA) was grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) containing 10 % fetal calf serum, streptomycin (100 μg/ml), penicillin (100 IU/ml) and fungizone (250 ng/ml) at 37°C in a humidified atmosphere of 95 % air and 5 % CO2.
Cell viability was assessed by WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay (Cell Counting Kit; Dojindo, Kumamoto, Japan) as previously described(Reference Ishiyama, Tominaga and Shiga15).
Enzyme-linked immunosorbent assay
HAEC were grown on a ninety-six-well plate and pretreated for 24 h with CS or FS (kindly provided by Ajinomoto Co., Kawasaki, Japan) at different concentrations (from 0·1 to 10 μm), or 40 μm-α-tocopherol (α-Toc) (positive control), or 10 μm-5-HT, -p-ca or -fa that represent the components of the structure of CS and FS (all from Sigma, St Louis, MO, USA), then stimulated with 25 mm-d-(+)-glucose (HG) (Sigma) for 4 h. Enzyme-linked immunoassay was used to assess VCAM-1 expression on HAEC as previously described(Reference Yoshida, Manabe and Terasawa16). MCP-1 protein level in the cell culture supernatant fraction was examined using a human MCP-1 Immunoassay kit (Biosource International, Camarillo, CA, USA) according to the manufacturer's protocol.
Glucose uptake by human aortic endothelial cells
Glucose uptake was performed as previously described(Reference Zou, Wang and Shen17) using a fluorescent d-glucose analogue 2-[N-(7-nitrobenx-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG; Invitrogen Life Technologies), used to monitor glucose uptake in living cells. HAEC were treated with 2-NBDG for 1 h in the simultaneous presence or absence of 10 μm-CS or -FS. Untreated HAEC were used as the control group. Fluorescence was observed using the Cytomics FC500 Flow Cytometry System (Beckman Coulter, Fullerton, CA, USA).
Monocyte adhesion and transmigration assay
The adhesion assay was performed as previously described(Reference Reddy, Hama and Grijalva18). HAEC were pretreated with 10 μm-CS, -FS, -5-HT, -p-ca or -fa or 40 μm-α-Toc for 24 h, then stimulated with HG for 4 h. Subsequently, 2′,7′-bis (2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (Molecular Probes, Eugene, OR, USA)-labelled U-937 (1 × 106 cells/ml, 100 μl) were added to each well, in the same media with HG in the presence or absence of the antioxidant used, and incubated at 37°C for 30 min. The same treatment was performed for the transmigration assay, using transwell membranes and adding labelled monocytes into the upper compartment as previously described(Reference Ryu, Hong and Maeng19). 2′,7′-Bis (2-carboxyethyl)-5(6)-carboxyfluorescein fluorescent intensity of adherent and transmigrated U-937 was quantified using the previously described method(Reference Chen, Chen and Lin20).
Isolation of mRNA and real-time PCR
HAEC were pretreated with 10 μm-CS or -FS for 24 h, then stimulated with HG for 3 h. Total RNA was isolated with the acid guanidinium phenol chloroform method using an Isogen kit (Nippon Gene, Tokyo, Japan). Primers were as follows: for VCAM-1, sense 5′-CCCTTGACCGGCTGGAGATT-3′, antisense 5′-CTGGGGGCAACATTGACATAAAGTG-3′; for MCP-1, sense 5′-CGCCTCCAGCATGAAAGTCT-3′, antisense 5′-GGAATGAAGGTGGCTGCTATG-3′; for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sense 5′-ACCACAGTCCATGCCATCAC-3′, antisense 5′-TCCACCACCCTGTTGCTGTA-3′. In order to quantify VCAM-1 and MCP-1 gene expression, the VCAM-1 and MCP-1 mRNA level was normalised by the total RNA content (μg/μl) and GAPDH mRNA level.
NF-κB activation assay
HAEC were treated with HG for 2 h in the simultaneous presence or absence of 10 μm-CS, 10 μm-FS, 10 μm-thenoyltrifluoroacetone (Aldrich, St Louis, MO, USA), a complex II inhibitor, 0·5 μm-m-chlorophenylhydrazone (Sigma), a mitochondrial membrane proton gradient inhibitor, or 100 μm-Mn(III)5,10,15,20-tetrakis(N-methyl-pyridinium-2-yl) porphyrin (Sigma), a synthetic cell-permeable Mn porphyrin-based superoxide dismutase mimetic. Subsequently, HAEC nuclear extraction was performed using a Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's protocol. NF-κB on nuclear extracts was detected using a TransAM NF-κB p65 Kit (Active Motif) according to the manufacturer's protocol.
Determination of intracellular reactive oxygen species
HAEC were stimulated with HG for 1 h in the simultaneous presence or absence of the compounds described for the adhesion and NF-κB assay. Intracellular reactive oxygen species (ROS) were detected using the fluorescent probe 2′,7′-dichlorofluorescin diacetate as described earlier(Reference Takahashi, Shibata and Niki21) and RedoxSensor Red CC-1 (Molecular Probes, Eugene, OR, USA). Mitochondria were labelled using the MitoTraker Green FM (Molecular Probes). The MitoTraker Green FM stain in conjunction with a RedoxSensor Red CC-1 stain was used to verify the distribution of the oxidised product, according to the manufacturer's protocol. Mitochondrial superoxide was detected using a MitoSOX mitochondrial superoxide indicator (Molecular Probes). Cells were observed using the FV1000 fluoview BX61 (Olympus, Center Valley, PA, USA).
HPLC analysis
HAEC were treated with 20 μm-CS or -FS for the specified periods (0, 24, 48, 72, 96 h). Untreated HAEC were used as the control group. Cells were then collected, and a sample was used for mitochondria extraction performed using a Mitochondria Isolation Kit (Pierce, Rockford, IL, USA) according to the manufacturer's protocol. Subsequently, CS and FS were extracted with methanol–PBS (4:1, v/v) and were measured by HPLC as described earlier(Reference Yoshida, Hayakawa and Habuchi22). In order to quantify the concentration, CS and FS levels were normalised by the total protein content (μmol/mg protein).
Data analysis
All data of at least three separate experiments were expressed as mean values with their standard errors. Two-way ANOVA was used to assess the differences between multiple groups using the GraphPad Prism 4 program (GraphPad Software, Inc., San Diego, CA, USA). Values of P < 0·05 were considered statistically significant.
Results
Effects of N-(p-coumaroyl)serotonin and N-feruloylserotonin on short-term high glucose-induced vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 and mRNA expression in human aortic endothelial cells
HG treatment for 4 h caused a significant four-fold increase in the surface expression of VCAM-1 protein (Fig. 2(A)) and a significant three-fold increase of MCP-1 protein release (Fig. 2(B)) in the cell culture supernatant fraction. Pretreatment of HAEC with CS and FS for 24 h decreased VCAM-1 expression and MCP-1 release in a concentration-dependent manner, with a significant inhibition at 5 and 10 μm. 40 μm-α-Toc had a similar effect to 10 μm-CS and -FS, while treatment with 10 μm-5-HT, -p-ca or -fa resulted in a non-significant reduction of VCAM-1 expression and MCP-1 release, which appeared to be significant only for 5-HT. These findings were further confirmed by mRNA expression analysis for VCAM-1, MCP-1 and GAPDH genes in HAEC by real-time PCR (Fig. 3(A)). Pretreatment of HAEC with 10 μm-CS or -FS for 24 h resulted in a significant decrease of HG-induced mRNA expression for VCAM-1 and MCP-1. The protection exerted by CS and FS was not the result of a prevention of cell glucose uptake, as shown by the flow cytometry analysis results. Cells treated with CS and FS with the addition of the fluorescent d-glucose analogue 2-NBDG exhibited the same fluorescence as that shown on cells treated with 2-NBDG alone (Fig. 3(B)).
Effects of N-(p-coumaroyl)serotonin (
) and N-feruloylserotonin (
) on short-term high-glucose (HG; 25 mm-d-(+)-glucose)-induced (A) vascular cell adhesion molecule-1 (VCAM-1) protein expression and (B) monocyte chemoattractant protein-1 (MCP-1) protein release, normalised to total cell protein (optical density arbitrary units/μg protein for VCAM-1 and ng/μg protein for MCP-1). C, control; α-Toc, α-tocopherol; 5-HT, serotonin; p-ca, p-coumaric acid; fa, ferulic acid. Values are means, with their standard errors represented by vertical bars. Mean value was significantly different from that for HG alone: *P < 0·05, **P < 0·01. ††† Mean value was significantly different from that for the control condition (P < 0·001).
Effects of N-(p-coumaroyl)serotonin (CS) and N-feruloylserotonin (FS) on short-term high-glucose (HG; 25 mm-d-(+)-glucose)-induced mRNA expression in human aortic endothelial cells (HAEC), and HAEC glucose uptake. (A) Vascular cell adhesion molecule-1 (VCAM-1; 
) and monocyte chemoattractant protein-1 (MCP-1; ) mRNA level, normalised by the total RNA content (μg/μl) and glyceraldehyde 3-phosphate dehydrogenase mRNA level. C, control; α-Toc, α-tocopherol. Values are means, with their standard errors represented by vertical bars. Mean value was significantly different from that for HG alone: *P < 0·05, **P < 0·01. †† Mean value was significantly different from that for the control condition (P < 0·01). (B) Cell glucose uptake, as shown by flow cytometry analysis. Trace a, control condition; trace b, 2-[N-(7-nitrobenx-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG); trace c, CS +2-NBDG; trace d, FS +2-NBDG.
Effects of N-(p-coumaroyl)serotonin and N-feruloylserotonin on short-term high glucose-induced monocyte adhesion and migration to human aortic endothelial cells
Incubation of HAEC with HG for 4 h caused an approximate three-fold increase in adhesion of U-937 compared with the adhesion of U-937 to control HAEC (Fig. 4(A)), and an approximately three-fold increase in transmigration of U-937 to the lower compartment compared with the transmigration of U-937 to control HAEC (Fig. 4(B)). Pretreatment of HAEC with 10 μm-CS or -FS for 24 h resulted in a significant decrease of HG-induced adhesion and migration of U-937 to HAEC. 40 μm-α-Toc had a comparable effect to 10 μm-CS or -FS, while treatment with 10 μm-5-HT, -p-ca or -fa resulted in a non-significant reduction of monocyte adhesion and migration.
Effects of N-(p-coumaroyl)serotonin () and N-feruloylserotonin () on short-term high-glucose (HG; 25 mm-d-(+)-glucose)-induced U-937 adhesion (A) and migration (B) to human aortic endothelial cells (HAEC). (A) 2′,7′-Bis (2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) fluorescent intensity of adherent cells. (B) BCECF-AM fluorescent intensity of migrated cells to the lower compartment. C, control; α-Toc, α-tocopherol; 5-HT, serotonin; p-ca, p-coumaric acid; fa, ferulic acid. Values are means, with their standard errors represented by vertical bars. ** Mean value was significantly different from that for HG alone (P < 0·01). ††† Mean value was significantly different from that for the control condition (P < 0·001).
Effect of N-(p-coumaroyl)serotonin and N-feruloylserotonin on short-term high glucose-induced NF-κB activation
HG treatment for 2 h induced a significant increase of the NF-κB binding activity of approximately 2·6-fold when compared with control HAEC (Fig. 5). This HG-induced effect was prevented by all the antioxidants used.
Effect of N-(p-coumaroyl)serotonin () and N-feruloylserotonin () on short-term high-glucose (HG; 25 mm-d-(+)-glucose)-induced NF-κB binding activity. C, control; CCCP, m-chlorophenylhydrazone; TTFA, thenoyltrifluoroacetone; MnTM2PyP, Mn(III)5,10,15,20-tetrakis(N-methyl-pyridinium-2-yl) porphyrin; α-Toc, α-tocopherol. Values are means, with their standard errors represented by vertical bars. Mean value was significantly different from that for HG alone: *P < 0·05, **P < 0·01. †† Mean value was significantly different from that for the control condition (P < 0·01).
Effect of N-(p-coumaroyl)serotonin and N-feruloylserotonin on short-term high glucose-induced intracellular reactive oxygen species
The HG-induced NF-κB activation on HAEC was preceded by a significant increase in the intracellular ROS levels (Fig. 6(A)) after treatment of HAEC with HG for 1 h, which was significantly reduced by CS and FS. 40 μm-α-Toc had a similar effect to 10 μm-CS and -FS, while treatment with 10 μm-p-ca or -fa resulted in a non-significant reduction of ROS fluorescence, which appeared to be significant only for 5-HT. The distribution of the oxidised product (Fig. 6(B)) showed red fluorescence of ROS in green mitochondria (lower images); HG-treated HAEC showed yellow areas because of co-localisation of green and red fluorescence as a result of ROS in mitochondria (HG, lower images). Co-localisation was not present in the control, or in any other treated group (lower images). These results were confirmed by the MitoSOX fluorescence, specific for mitochondrial superoxide, that was highly present on HG-treated HAEC (HG, upper image), but not relevant in any other treated group (upper images).
Effect of N-(p-coumaroyl)serotonin (CS; ) and N-feruloylserotonin (FS; ) on short-term high-glucose (HG; 25 mm-d-(+)-glucose)-induced intracellular reactive oxygen species (ROS). (A) Intracellular ROS assessed by the fluorescence intensity of dichlorofluorescin (DCF) emission. C, control; α-Toc, α-tocopherol; 5-HT, serotonin; p-ca, p-coumaric acid; fa, ferulic acid. Values are means, with their standard errors represented by vertical bars. Mean value was significantly different from that for HG alone: *P < 0·05, **P < 0·01. †† Mean value was significantly different from that for the control condition (P < 0·01). (B) Intracellular ROS detected by RedoxSensor Red CC-1 (red fluorescence, lower images), mitochondria labelled by Green-Fluorescent MitoTraker Green FM (green fluorescence, lower images) and mitochondrial superoxide detected by MitoSOX (green fluorescence, upper images). CCCP, m-chlorophenylhydrazone; TTFA, thenoyltrifluoroacetone; MnTM2PyP, Mn(III)5,10,15,20-tetrakis(N-methyl-pyridinium-2-yl) porphyrin.
N-(p-coumaroyl)serotonin and N-feruloylserotonin concentration inside human aortic endothelial cells and mitochondria
Treatment with 20 μm-CS from 0 to 96 h resulted in a time-dependent increase of CS concentration inside the cells and the mitochondria; the increase was significant after 48 h inside the cells (6·9 μmol CS/mg protein) and after 72 h inside the mitochondria (4·6 μmol CS/mg protein) (Fig. 7). A similar pattern was obtained for FS detection (data not shown).
N-(p-coumaroyl)serotonin (CS) concentration inside whole human aortic endothelial cells (–●–) and mitochondria (–○–). CS level was normalised by the total protein content (μmol/mg protein). Values are means, with their standard errors represented by vertical bars. Mean value was significantly different from that at time 0: *P < 0·05, **P < 0·01, ***P < 0·001.
Discussion
CS and FS, two serotonin conjugates, have been identified from safflower seeds(Reference Zhang, Nagatsu and Watanabe3), konnyaku(Reference Niwa, Murakami and Ohtake5) and Japanese barnyard millet(Reference Watanabe6). They exhibit antioxidant and anti-inflammatory activities(Reference Zhang, Nagatsu and Watanabe3–Reference Kumarasamy, Middleton and Reid7, Reference Choi, Lee and Kim10), together with many species of the genus Centaurea, traditionally used for their antibacterial activity in the treatment of various aliments(Reference Kumarasamy, Middleton and Reid7), such as cornflower (Centaurea cyanus L.)(Reference Sarker, Laird and Nahar8) and sweet sultan (Centaurea moschata L.)(Reference Sarker, Savchenko and Whiting9). In addition to these already mentioned plants and their effects, CS and FS have been detected in some vegetables, such as paprika and green onions, which contain CS, and tomatoes, cherry tomatoes, hot peppers, paprika, lettuce, Chinese cabbage, chicory and spinach, which contain FS(Reference Ly, Kang and Choi4). Finally, maize bran, which is an important byproduct of the maize dry milling industry, is rich in several functional lipid constituents, including serotonin hydroxycinnamic acids, possessing antioxidant and anti-melanogenic activities(Reference Choi, Lee and Kim10). Although it has been reported that CS and FS were protective against LDL oxidation and atherogenesis in experimental animals and in human subjects(Reference Koyama, Kuribayashi and Seki2, Reference Koyama, Suzuki and Furukawa13), to the best of our knowledge, cell-based studies on their action against endothelial damage have not been reported.
The migration of circulating monocytes into the subendothelial space is one of the crucial events in early atherogenesis. VCAM-1 and MCP-1 are well-known factors involved in this process; VCAM-1 mediates the adhesion of leucocytes to activated endothelium(Reference Albelda, Smith and Ward23), MCP-1 is a powerful monocyte chemoattractant, both in vivo and in vitro (Reference Gerszten, Garcia-Zepeda and Lim24), and both are expressed by endothelial cells in early atherosclerotic lesions and involved in monocyte adhesion and recruitment to early lesions(Reference Piga, Naito and Kokura14). In accordance with the protective effect of CS and FS against atherogenesis as previously reported in vivo (Reference Koyama, Kuribayashi and Seki2, Reference Koyama, Suzuki and Furukawa13), we found that the pretreatment of HAEC with CS or FS reduced the HG-stimulated HAEC expression of VCAM-1 and MCP-1 in a concentration-dependent manner, as well as the adhesion and migration of U-937. We also compared these inhibitory effects with the effect of 40 μm-α-Toc, which is the level achieved in plasma by consuming 133·4 mg (200 IU) α-Toc supplements/d(Reference Kumarasamy, Middleton and Reid7, Reference Kaliora, Dedoussis and Schmidt25, Reference Liu, Zubik and Collins26), and with the effect of 5-HT, p-ca and fa, which represent the components of the structure of CS and FS; CS and FS protection was higher than that of 5-HT, p-ca, and fa alone in the experimental condition used, suggesting that their inhibitory effect may be due to both 5-HT and p-ca or fa moieties acting together, and was also comparable with the protective effect of 40 μm-α-Toc, used as a positive control. These treatments did not affect cell viability (data not shown). In addition, similar results were obtained with the simultaneous treatment of HAEC with CS and FS plus HG without 24 h CS or FS pretreatment (data not shown), suggesting that pretreatment with CS and FS is not an essential requirement on the inhibitory effect of CS and FS against VCAM-1 and MCP-1 increase. Our observations are novel and provide a potential mechanism by which CS and FS reduce the risk of atherosclerosis in vivo.
Because NF-κB is activated by reactive oxygen species, and VCAM-1 and MCP-1 expression is also regulated by NF-κB(Reference Piga, Naito and Kokura14, Reference Collins, Read and Neish27), the radical scavenging activity of CS and FS may contribute to the inhibition of the expression of VCAM-1 and MCP-1. Furthermore, previous reports(Reference Nishikawa, Edelstein and Du28) showed a hyperglycaemia-increased activation of NF-κB that was prevented by thenoyltrifluoroacetone, m-chlorophenylhydrazone, and by an overexpression of the Mn superoxide dismutase, the mitochondrial form of this antioxidant enzyme. This suggests that the superoxide is the reactive oxygen radical produced by this mechanism, that the mitochondria is the site of hyperglycaemia-induced ROS, and that hyperglycaemia activates NF-κB through a ROS-dependent pathway. Therefore, we tested the effect of CS and FS in comparison with the effect of Mn(III)5,10,15,20-tetrakis(N-methyl-pyridinium-2-yl) porphyrin, thenoyltrifluoroacetone and m-chlorophenylhydrazone on short-term HG-induced NF-κB and ROS in HAEC to clarify and compare their ability to penetrate inside the mitochondria and their role against the superoxide; furthermore, the distribution of the oxidised products was also determined. Each compound completely inhibited NF-κB activation and ROS production; the distribution of the mitochondrial superoxide revealed fluorescence of the mitochondrial superoxide only in HG-treated HAEC. These results indicated that CS and FS are acting on the superoxide inside the mitochondria, and that their scavenger activity against mitochondrial superoxide overproduction was effective in preventing the consequent activation of NF-κB. In addition, the ability of CS and FS to reduce ROS was higher than that of 5-HT, p-ca, and fa alone(Reference Kaliora, Dedoussis and Schmidt25, Reference Kajimoto, Ikebe and Yoshida29–Reference Rozek, Achaerandio and Almajano31) in the experimental conditions used, suggesting that CS and FS antioxidant activity may be due to both 5-HT and p-ca or fa moieties acting together. In addition, ROS-scavenging activity by CS and FS was comparable with that exerted by 40 μm-α-Toc.
As a consequence of the biological activities of these two serotonin derivatives, it is possible that they are acting as direct scavengers of the superoxide radical or by scavenging the peroxynitrite generated by the reaction of the superoxide with NO. Indeed, the intracellular ROS fluorescence detected using Red CC-1 and 2′,7′-dichlorofluorescin diacetate, which are not specific for the superoxide, could also be due to the presence of peroxynitrite. The consequent dangerous peroxynitrite generation is then neutralised by the scavenging activity of CS and FS.
Since MitoSOX results confirmed an early uptake of CS and FS into the mitochondria, we quantified the concentration of both compounds inside the HAEC and their mitochondria by HPLC. Even though the presence of CS and FS inside the cells was not detectable by HPLC after 10 μm treatment and 1–4 h incubation, the treatment of HAEC with 20 μm-CS or -FS from 24 to 96 h revealed a time-dependent increase of CS or FS content inside the cells and mitochondria, the increases significant after 48 h although detectable from 24 h. These results may suggest a potential role of CS and FS as protective agents against prolonged insults and may explain their protective effect in vivo due to the stability of their molecules, which do not show a time-dependent modification, transformation or degradation.
In conclusion, using a cell-based approach, the present study suggested a serotonin derivative mechanism against the increase of ROS-dependent VCAM-1 and MCP-1, due to the derivatives' strong antioxidant properties and scavenger abilities. The presence of CS and FS in several wild-growing plants and their seeds, as well as in grains and vegetables, suggests that they could be useful sources of natural antioxidants, and that they could be helpful in reducing the incidence of atherosclerosis and CVD especially in Western society where it is particularly high(Reference Tunstall-Pedoe, Kuulasmaa and Amouyel1). Furthermore, they could also be taken into consideration as an alternative and/or preventive remedy against ROS-related dysfunctions.
Acknowledgements
The authors are employees of the Kyoto Prefectural University of Medicine and the work described in the present article has been funded by a Grant-in-Aid for Scientific Research (15390178 TY) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, from the Bio-oriented Technology Research Advancement Institution and from the Ministry of Agriculture, Forestry and Fisheries of Japan. All the authors designed the study. R. P. conducted the experiments and drafted the paper. Y. N., S. K., O. H. and T. Y. provided helpful technical advice and support in all the experiments carried out, and also supervised the entire work.
The authors are grateful to Professor Etsuo Niki, Professor Yoshiro Saito and Professor Yasukazu Yoshida, Human Stress Signal Research Center, AIST Kansai, Japan, for their assistance on HPLC analysis and critical discussion of data.
The authors declare that they have no other conflict of interest.
