S‑1‑Propenylcysteine, a sulfur compound in aged garlic extract, alleviates cold‑induced reduction in peripheral blood flow in rat via activation of the AMPK/eNOS/NO pathway
- Authors:
- Published online on: July 7, 2020 https://doi.org/10.3892/etm.2020.8969
- Pages: 2815-2821
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Copyright: © Ushijima et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Blood circulation plays a fundamental role in the body homeostasis by supporting the critical functions including the transport of water, oxygen, nutrients, and hormones as well as carbon dioxide and metabolic waste from cells for removal.
With the change of modern lifestyle, people suffer from chilliness due to mental stress (1) and excessive use of air-conditioning systems (2). Raynaud's phenomenon (RP) is a disease caused by vasoconstriction of cutaneous arterioles induced by cold exposure and mental stress and is accompanied by such characteristic symptoms as cold sensation in the limbs, pain, numbness, swelling, and digital ulcers (3,4). Current pharmacological treatments including calcium channel blockers, phosphodiesterase type 5 inhibitors, intravenous prostanoids, and topical nitrates fail to completely control RP and are not well tolerated by some patients (4).
Aged garlic extract (AGE) is a unique garlic product prepared through long extraction processes with water-ethanol for more than 10 months (5). During this period, many harsh and strong odor substances (i.e., allicin) in raw garlic are converted into non-irritating water-soluble sulfur-containing amino acids such as S-allylcysteine (SAC), S-1-propenylcysteine (S1PC), and S-allylmercaptocysteine (SAMC) (6,7).
Previous studies have demonstrated the beneficial effect of AGE on hypertension (8-12), atherosclerosis (13-15), metabolic syndrome (16), and gingivitis (17). Furthermore, recent studies have revealed that AGE and its constituents, SAC and S1PC, exert such desirable effects as anti-hypertension (18-22), cardioprotection (23), antioxidation (24-27), antiaging (27,28), immunomodulation (29-31), anti-fatigue (32,33), anti-stress (34,35), anti-inflammation (36-39), and liver protection (40).
Several previous studies show that AGE improves the peripheral circulatory disturbances. One clinical study using thermography demonstrated that AGE supplementation increases microcirculation and improves a variety of symptoms in patients (41). In another clinical study, AGE has been shown to enhance cutaneous microcirculation in patients with the increased risk of cardiovascular events (42). In addition, an animal study demonstrated that AGE can reverse the decrease in the rectal temperature of mice induced by cooling (32,43). Furthermore, administration of S1PC, a characteristic constituent of AGE increased the blood flow of tail skin in spontaneously hypertensive rats (SHR) (21).
The above reports indicate that AGE is effective in improving the peripheral circulation in both animal and human. At present, however, the mechanism whereby AGE improves the circulation remains unclear. Hence, this study was conducted with the aim to identify the active constituent of AGE and to clarify its mechanism of action using the model of cold-induced reduction in the tail blood flow of rat.
Materials and methods
Chemicals
AGE was prepared from cloves of garlic (Allium sativum L.) through the process of rinsing with purified water, slicing, soaking in ethanol 20-50% (v/v), and extraction/aging for more than 10 months (5). S1PC was isolated and purified from AGE using preparative high-performance liquid chromatography (HPLC), as previously reported (7). SAC and SAMC were synthesized according to previous methods (7). Chemicals for synthesis of compounds and post-column HPLC detection reagents were obtained from Tokyo Chemical Industry. Solvents for HPLC were purchased from Wako Pure Chemicals Industry. Authentic sulfur compounds for identification were synthesized and purified according to the previous report (7).
HPLC analysis
HPLC analysis was performed by post-column HPLC using a NEXERA X2 system (Shimadzu, Kyoto, Japan) according to the previous report (7). The column utilized for separation was a Cadenza CD-C18 column (2.0 x 150 mm, 3 µm; Imtakt Corporation). The sulfur compounds were derivatized with the hexaiodoplatinate reagents (Tokyo Chemical Industry) and detected at 500 nm absorbance using diode array detector.
Animals and test substances
Specific pathogen-free male Wistar rats were obtained at the age of 7 weeks from Japan SLC, Inc (Shizuoka, Japan). Rats were kept at 23±3˚C and 50±10% humidity under a 12-h light-dark cycle (light 7:00 a.m.-7:00 p.m.), with free access to commercially available hard feed (CE-2) and water, until the experimental use at 10 to 14-weeks of age. AGE was diluted at 0.2 g/ml with distilled water (DW). SAC, S1PC, and SAMC were dissolved in DW. All test substances (AGE, SAC, S1PC, and SAMC) were administered to rats in a volume of 10 ml/kg BW using Teflon feeding needles. The control group was orally administered DW in a volume of 10 ml/kg BW. Animal experiments were approved by the Animal Care and Use Committee of Wakunaga Pharmaceutical Co., Ltd. (approval no. 258). This investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication, 8th edition, 2011).
Cold-induced model of reduced tail blood flow
The cold-induced model of peripheral circulation disorder was designed based on the rat cooling RP (44) and cold blood stasis syndrome models (45,46). After being acclimatized to the experiment room at 23˚C for at least 30 min, rats were examined for skin blood flow before treatment and then orally administered test substances. Two hours later, the rats were placed in restrictive cages (KN-468-B; Natsume) for cooling and submersed in tank filled with 15˚C water up to the xiphoid processes for 10 min. In the case of non-cooling experiments, rats were placed in restrictive cages for 10 min without cooling. After being returned to rearing cages, the rats were examined for the tail blood flows 1 h after cooling.
Measurement of tail blood flow
The tail blood flow of rats was measured by using a contact laser Doppler blood flow meter (FLO-C1; Omegawave). Each rat was placed in a holder under anesthesia with isoflurane (1 l/min) and its tail blood flow was examined for 3 min. The probe for the measurement was attached 5 cm apart from the base of the tail. Rats were cooled for 10 min at 2 h after administering AGE (2 g/kg BW; n=10) or S1PC (6.5 mg/kg BW; n=9), and then measured for their tail blood flows at 1, 2 and 3 h after cooling. For the comparison of the effect of three sulfur constituents, rats were orally administered SAC (7.9 mg/kg BW), SAMC (1.3 mg/kg BW), or S1PC (0.26, 1.3 and 6.5 mg/kg BW), followed by cooling for 10 min at 2 h later. The tail blood flow was measured at 1 h after cooling (n=10). The dose of SAC (7.9 mg/kg BW), SAMC (1.3 mg/kg BW), and S1PC (6.5 mg/kg BW) was equivalent to the amount contained in AGE at 2 g/kg BW. In another study, rats were also measured for their tail blood flow without cooling after oral administration of AGE (2 g/kg BW) or S1PC (6.5 mg/kg BW; n=6).
Measurements of plasma NOx level and vascular NO-related phosphorylation
Rats were cooled for 10 min at 2 h after administration of S1PC (6.5 mg/kg BW) or DW (control) and then anesthetized with isoflurane (1 l/min) to collect the blood from the orbital vein at 1 h after cooling. Another group of rats was administered DW and did not receive the cooling treatment (non-cooling control). After centrifugation of blood samples at 1,500 x g for 15 min at 4˚C, plasma samples were obtained and stored at -80˚C until analysis. Thoracic aortas of rats were also isolated, frozen by liquid nitrogen, and stored at -80˚C until analysis. The plasma content of NOx was determined by using a NOx colorimetric assay kit (Cayman Chemical Company) according to the manufacturer's instructions. Rat thoracic aortas were homogenized using the Multi-Beads shocker (Yasui Kikai) and lysed by a RIPA lysis buffer (Merck KGaA) containing Halt protease and phosphatase inhibitor (Thermo Fisher Scientific, Inc.). Western blot analysis of the lysates was performed as previously described (40). In brief, individual proteins were separated by SDS-PAGE and immunoblotted with an anti-phospho-eNOS (Ser1177; Cell Signaling Technology, Inc.), anti-eNOS (Santa Cruz Biotechnology, Inc.), anti-phospho-AMPKα (Thr172; Cell Signaling Technology, Inc.), anti-AMPKα (ProteinTech Group, Inc.), and anti-GAPDH antibody (Wako). Immunoreactive bands were visualized and analyzed on a V3 Western Workflow (Bio-Rad Laboratories, Inc.) using Image Lab™ software.
Statistical analysis
The data were expressed as mean ± SEM. Statistically significant differences between test substance-treated and control DW-treated groups were determined using Student's t-test or one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using WinSTAT (R. Fitch Software).
Results
Characterization of hydrophilic sulfur compounds in AGE
AGE used in this study was analyzed by post-column HPLC using hexaiodoplatinate reagent for sulfur-specific detection (7). This analysis detected and identified 11 hydrophilic sulfur constituents such as S-methylcysteine, SAC, S1PC, and SAMC which are formed in AGE through the aging process (7) (Fig. 1 and Table I).
 | Table ICharacterization of hydrophilic sulfur compounds in aged garlic extract by post-column HPLC chromatogram. |
Effects of AGE and its constituents on tail blood flow
After cooling (15˚C, 10 min), the tail blood flow of DW-pretreated control rats decreased 53% at 1 h, as compared to the non-cooling level (P<0.01), and then returned almost to the initial level at 3 h (Fig. 2). On the other hand, the tail blood flow of AGE (2 g/kg BW)-pretreated group was significantly (P<0.01) higher than that of DW-pretreated control group after cooling and returned to almost initial non-cooling level (Fig. 2).
The effect of AGE constituents, SAC, S1PC, and SAMC, on the blood flow was also examined. As shown in Fig. 3, pretreatment with S1PC (6.5 mg/kg BW) significantly (P<0.05) improved the reduction in the tail blood flow compared to the pretreatment with DW (control) at 1 h after cooling. However, pretreatment with SAC or SAMC showed no significant improvement. As shown in Fig. 4, the effect of S1PC is dose-dependent: and the dose of S1PC 6.5 mg/kg BW produced a larger increase of tail blood flow. Fig. 5 showed the time course pattern of tail blood flow in the S1PC (6.5 mg/kg BW)-pretreated group after cooling. At 1 h after cooling, tail blood flow was significantly higher (P<0.05) in the S1PC-pretreated group than in the DW-pretreated control group. Thereafter, no significant difference was observed in the tail blood flow between the two groups. Both S1PC and AGE pretreatment had no significant effect on the tail blood flow of non-cooled rats (data not shown).
Effect of S1PC on NOx concentration in plasma
NO is known to trigger the vasodilation by initiating the signaling cascade (47). NO is a gas with a very short plasma half life of only a few seconds and thus its level is difficult to measure. It is a common practice to estimate the NO production by measuring its stable metabolites (NOx). As shown in Table Ⅱ, the cooling of rat significantly (P<0.01) increased the plasma NOx concentration as indicated by the comparison between the two groups of rats with and without cold treatment. Moreover, pretreatment of rats with S1PC (6.5 mg/kg BW) further increased the plasma NOx level (P<0.05), compared to non-pretreated rats.
Effect of S1PC on eNOS and AMPK phosphorylation in aorta
Fig. 6 shows the effect of S1PC on the level of phosphorylated eNOS and AMPK in aorta that are key regulatory molecules of NO production in the vascular endothelium. By western blot analysis, the phosphorylation level of both eNOS and AMPK was found to be up-regulated by cooling. Additionally, pretreatment of rat with S1PC (6.5 mg/kg BW) significantly increased the phosphorylation of eNOS and AMPK compared to pretreatment with DW.
Discussion
AGE reportedly promotes the recovery of rectal temperature decline in mice induced by cooling, and its effect is more potent than that of raw or other garlic preparations (32). Recently it was reported that AGE and S1PC, a constituent of AGE, significantly increase the tail blood flow of SHR (21). At present, however, the underlying mechanism of AGE action has not been fully elucidated.
In this study, we examined the effects of AGE and its three characteristic sulfur compounds SAC, S1PC, and SAMC on the peripheral blood circulation by using a cold-treated rat model; the tail blood flow of rat was reduced by cooling, and the effect of pretreatment with AGE and these three constituents was examined in terms of the recovery of the decreased blood flow. We found that pretreatment with AGE or S1PC significantly counteracted the reduction of tail blood flow of the rat caused by cooling. The effect of S1PC is dose-dependent with the maximal dose of 6.5 mg/kg BW and specific in the sense that other constituents of AGE, SAC and SAMC had no effect on the reduction of tail blood flow. These results suggested that S1PC is a major active constituent responsible for the effect of AGE to improve the peripheral blood circulation.
The skin blood flow is regulated by both the sympathetic vasoconstrictor system and nonadrenergic sympathetic active vasodilator system to maintain body temperature (48,49). Under the cold conditions, the vasoconstriction of peripheral artery is induced by both activation of sympathetic reflex and reduction of locally produced NO (50), leading to poor circulation and low body temperature. In addition, Koganezawa et al (51) found that cooling of the rat skin stimulates presynaptic P2 purinoceptors on sympathetic nerve terminals and facilitates the release of noradrenaline, thereby inducing contraction of skin blood vessels via the activation of α1- and α2-adrenoceptors. Thus it was conceivable that S1PC improves the blood flow of cooled rat by inhibiting noradrenaline signaling and/or the enhancement of NO production. However, Takashima et al (52) reported that S1PC had no effect on noradrenaline-induced contraction in isolated rat blood vessels. Thus it is more likely that S1PC improves the blood flow by acting on NO production system but not on the pathway of noradrenalin-triggered vasoconstriction.
The NO production system has a critical role for vasodilation; NO is produced by eNOS on vascular endothelium, that increases cGMP levels via activating soluble guanylyl cyclase present in smooth muscle cells and thereby causes vascular smooth muscle relaxation (53,54). In addition, it was shown that NO was involved in the recovery of the reduced human skin blood flow after cooling (50). Furthermore, a previous study reported that the NO synthesis inhibitor, L-NAME, delayed the recovery of tail skin temperature of mice decreased by cooling (55). Previously, AGE was shown to increase NO production in endothelial cells (52,56). In this study, the cooling of rats significantly increased the plasma NOx concentration (Table II). We also showed that additional pretreatment with S1PC further increased the concentration of NO metabolites in the plasma of cooled rats when compared to DW-pretreated control (Table II). These results suggested that S1PC improved the reduction of tail blood flow of cooled rats via enhancing NO production. It was reported that both low temperature and shear stress induce the activation of eNOS by activating AMPK (57,58). AMPK is known to phosphorylate the specific serine residue (Ser1177) of eNOS that is essential for its activation (58,59). It was also shown that AGE activated AMPK in both diabetic model TSOD mice (39), and arteriosclerotic model ApoE-KO mice (36). In addition, S1PC enhanced the AMPK activity in mouse spleen cells in vitro (37). In this study, the phosphorylation level of both eNOS and AMPK was up-regulated by cooling of rats (Fig. 6). The additional pretreatment with S1PC further increased the phosphorylation of eNOS and AMPK in the aorta of cooled rats, compared to DW-pretreated control (Fig. 6). On the other hand, S1PC did not affect the normal tail blood flow of non-cooled rats. These results suggest that S1PC enhances NO production induced by the low temperature and shear stress through the activation of AMPK, leading to improvement of peripheral circulatory disturbance.
In summary, this study showed that pretreatment with S1PC alleviated the cold-induced reduction of tail blood flow in rat by increasing the NOx concentration in plasma, and the phosphorylation of eNOS and AMPK in aorta. The findings suggest that S1PC may be a useful agent for the treatment of peripheral circulatory disorders including RP and poor circulation caused by smoking, aging, and stress.
Acknowledgements
The authors thank Dr Takami Oka of Wakunaga Pharmaceutical Co., Ltd. for his helpful advice, encouragement, and critical reading of the manuscript.
Funding
No funding was received.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Authors' contributions
MU, KK, and JIS designed the experiments, and MU performed blood flow measurement, and was a major contributor in writing the manuscript. KK performed measurements of plasma NOx level. JIS performed measurements of vascular NO-related phosphorylation. All authors have read and approved the final manuscript.
Ethics approval and consent to participate
Animal experiments were approved by the Wakunaga Pharmaceutical Co., Ltd. Institutional Animal Care and Use Committee (approval no. 258). This investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication, 8th edition, 2011).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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