During embryogenesis, retinoids, the metabolic products of carotenoids, play an essential role in the morphogenesis of the cardiovascular system. This role is mediated through the activation of the retinoic acid receptor (RAR) involved in signal transduction pathways regulating embryonic development, tissue homeostasis, and cellular differentiation and proliferation. However, previous studies have shown that retinoids have a major role in the cardiac remodeling process in hypertensive rats with myocardial infarction [1
Carotenoids; plant pigments are bioconverted to retinol then to retinoic acid (RA) first in the intestine, then the liver, and finally in target cells. RA is vital for modulating a wide range of biological processes such as cell differentiation and proliferation, vision, bone formation, metabolism, and immunological processes [2
]. RA is a potent transcriptional regulator and a natural ligand for the retinoic acid receptor (RAR) and retinoid X receptors (RXRs), which are DNA-binding transcriptional regulators. These receptors form homodimers (RXR/RXR) and heterodimers (RXR/RAR), which directly activate gene transcription, by binding to specific RA response elements in target gene promoter regions [3
]. Therefore the investigation and development of retinoid derivatives as drugs that target RARs and RXRs are very promising for the treatment of a multitude of ailments [4
Teodoresco; unicellular marine phytoplankton that belong to the phylum Chlorophyta [5
] are considered to be the richest natural producer of massive carotenoids. However, β-carotene, pro-vitamin A, remains the major natural product harvested from D. salina
(up to 1% dry weight). D. salina
exhibited the growth inhibition activity and proapoptotic effects on human colon cancer cell lines, many types of cancer, and degenerative diseases in vitro and in vivo [6
] probably due to their antioxidant and anti-inflammatory activities. The previous study held in our lab revealed a promising curative and prophylactic efficacy of D. salina
against cardiac dysfunction in senile rats [7
], which was an urge towards investigating the phytochemical constituent underlying this effect.
Hence, the present study aimed to investigate the beneficial effect of zeaxanthin heneicosylate (ZH); a major carotenoid isolated from D. salina on cardiac dysfunction associated with d-galactose (d-GAL)-induced aging in rats. To achieve this aim, electrocardiographs were recorded as well as assessment of serum cardiac function viz., homocysteine (HS), creatinine kinase isoenzyme (CK-MB), lactate dehydrogenase (LDH) and glucose trasporter 4 (GLUT-4); cardiac oxidative stress biomarkers viz., superoxide dismutase (SOD) and cardiac inflammatory mediators viz., inducible nitric oxide synthetase (iNOS) and interleukin-6 (IL-6) were all performed for both treated and untreated groups. Histopathological changes in the cardiac tissue were recorded. Moreover, retinoid acid receptor alpha (RAR-α) gene expression was estimated in cardiac tissues.
microalgae is considered a good candidate for promoting cardiovascular activity according to a previous work performed in our laboratory [8
]. The carotenoid rich fraction of D. salina
showed high efficacy in modulating d
-GAL induced cardiac dysfunction which promoted for further investigation of its phytoconstituents. Chromatographic analysis was performed resulting in the isolation of an orange amorphous powder which was identified according to spectral analysis as ZH (Figure 1
) which was confirmed through comparison with previously reported spectral data [10
]. Zeaxanthin, one of the most common carotenoids found in nature, is an anti-oxidant that accumulates in the retina of the human eye and protect the retinal structure from light-induced damage. It has been observed that consumption of diets with higher levels of zeaxanthin are accompanied by a low incidence of eye diseases such as age-related macular diseases, cataract, and diabetic retinopathy [11
]. Currently, the possible role of ZH on cardiac remodeling process in elderly rats was investigated.
Age-associated cardiac dysfunction was associated with dramatic changes in the ECG pattern and a prominent increase in the serum cardiac biomarkers levels as well as an elevation in cardiac oxidative stress and inflammatory mediators. Moreover, histopathological cardiac examination revealed prominent changes in cardiac tissue architecture. ECG pattern showed an irregular rhythm of heartbeats, depressed ST height, negative T waves as well as elevated PR and QRS intervals in respect to normal rats which is an indication of myocardial disease [12
]. In addition, heart rate significantly was elevated together with an increase in serum levels of cardiac function-relevant markers viz.
HS, CK-MK and LDH and decrease in the cardiac GLUT-4 content as compared to the control group.
Previous studies have shown that age-related changes in impulse propagation may be related to abnormalities in the pattern of ventricular activation [13
]. Recent data has suggested that alterations in heart rhythm intervals during aging may be associated with fibrosis and hypertrophy [14
In accordance with other studies, induction of age-associated cardiac dysfunction by d
-GAL showed a prominent elevation in cardiac levels of IL-6, iNOS, SOD and NF-κB. Aging is extensively associated with an imbalance between reactive oxygen species (ROS) production on one hand and antioxidant activities and NO bioactivity on the other [16
]. Furthermore, inflammatory cytokines such as IL-6 is involved in proinflammatory signaling in the aging cardiovascular system. IL-6 is increased in aged myocardium which promotes myocardial damage and matrix remodeling, including collagen deposition [17
]. Previously, it has been shown that the activity of myocardial NF-kB is increased in elder rats, which is reported to be associated with impairment of cardiomyocyte relaxation [18
Zeaxanthin heneicosylate isolated from D. salina
exhibited a satisfactory safety profile in the acute toxicity test where the experimental animals survived a single oral dose of up to 1 g/kg. Furthermore, ZH showed a significant improvement in ECG pattern, ST height, T wave and PR interval. It also restored the heart rate and QRS interval to normal range. Serum levels of HS, CK-MK and LDH showed a significant decline as well as cardiac GLUT-4 content. Additionally, ZH succeeded to decrease the cardiac level of IL-6 and iNOS and restore the cardiac SOD content to nearly the normal value and ameliorate the cardiac NF-κB level as compared to d
-GAL treated group. It has been reported that direct inhibition of NF-κB effectively inhibits cardiac hypertrophy and cardiac dysfunction associated with aging [20
]. Furthermore, the histopathological examination emphasized the obtained results where the cardiac tissue isolated from ZH treated rats showed minimal pathological alterations with respect to that isolated from normal ones. Moreover, ZH had the ability to restore the elevated levels of ALT, AST, urea and creatinine as compared to the d
-GAL injected rats. These results indicate that zeaxanthin may have a hepatoprotective role in aged rats in accordance with Xiao et al. (2014) [21
], a study that has been performed on an alcoholic fatty liver disease model and indicates as well that it protects the kidneys from the detrimental consequences of aging. There were no behavioral alterations in the ZH treatment group which further assures the safety of ZH. A number of studies investigated the safety of zeaxanthin and concluded that it shows a considerable amount of safety data based on regulatory studies. Subchronic studies with mice and rats receiving beadlet formulations of high purity synthetic zeaxanthin in the diet at dosages up to 1000 mg/kg body weight/day, and in dogs at over 400 mg/kg body weight/day, produced no adverse effects or histopathological changes. Zeaxanthin did not cause any signs of fetal toxicity or teratogenicity in rats or rabbits at dosages up to 1000 or 400 mg/kg bw/day, respectively. A 52-week chronic oral study in Cynomolgus monkeys at doses of 0.2 and 20 mg/kg bw/day, mainly designed to assess accumulation and effects in primate eyes, showed no adverse effects. In a rat two-generation study, the no-observed-adverse-effect-level (NOAEL) was 150 mg/kg bw/day. In 2012, this dosage was used by EFSA (NDA Panel), in association with a 200-fold safety factor, to propose an Acceptable Daily Intake equivalent to 53 mg/day for a 70 kg adult. The requested use level of 2 mg/day was ratified by the EU Commission [22
As far as we can find, there are three prospective describing the metabolic pathway of zeaxanthin in the mammalian body. The first postulation assumes that upon intestinal absorption zeaxanthin is incorporated in chylomicrons which are transferred to the liver, where they can be either stored or re-secreted into the circulation in association with lipoproteins [23
]. The second approach suggests a comparative pathway to that of beta-carotene and the potential oxidative cleavage of zeaxanthin by β-carotene-15,15’-oxygenase (BCO1) or β-carotene-9’,10’-oxygenase (BCO2) into the apo-oxidation analogues of retinol and retinoic acid [2
]. Finally, the third assumption involves an isomerization or oxidation at the position 3’which was based on the detection of meso-zeaxanthin and 3′-dehydro-lutein, respectively in human serum after supplementation with either lutein or zeaxanthin [24
] (Figure 7
In the course of investigating the effect on retinoic acid receptors, the oxidative metabolic analogues of retinoic acid; 15-apo and 9’-apozeaxanthenoic acids were studied for their affinity for RAR-α and RXR. Molecular docking of the 15-apo-carboxylic analogue of zeaxanthin showed high affinities towards RAR and RXR. The assumption that the pharmacological effect of ZH is mediated through the interaction with retinoid receptors was further confirmed through the evaluation of RAR-α gene expression which revealed a marked increase in the group treated with ZH as compared to the non-treated senile rats.
It has previously been shown that RA supplementation attenuates cardiac remodeling after experimental MI in rats [1
] where RAR which are abundantly expressed in cardiomyocytes, were activated through heterodimerization with RXRs [25
]. Studies showed that activation of RAR/RXR signaling prevented oxidative stress and apoptosis, in both neonatal and adult cardiomyocytes [26
]. Recently, it has been reported that activation of RAR signaling prevented diastolic dysfunction, through inhibition of intracellular ROS generation and NF-κB signaling-mediated inflammatory responses [27
These results proposed that RAR-mediated signaling has a major role in modulating cardiac oxidative stress due to pathological stimuli, which serves as an important mechanism in the development of diastolic dysfunction and heart failure. Experimental studies suggest that RA and similar ligands suppress both morphological alterations and changes in gene expression associated with hypertrophy which explains the effect of ZH [28
]. Most studies that relate the role of RAR/RXR in the regulation of adult heart function have used RAR selective ligands or antagonists, due to heart malformation and poor survivability of genetic models of RAR deletion [29
The findings of the current study are in line with the study by Iribarren et al., which revealed that the uptake of zeaxanthin was inversely related to the incidence of atherosclerosis [30
]. Voutilainen et al. also pointed out that there is a positive correlation between the higher intake of fruits and vegetables rich in carotenoids and the prevention of morbidity and mortality with relation to CVD [31
]. On the contrary, Bonds et al. reported that supplementation of zeaxanthin in addition to the daily intake of minerals and vitamins did not reduce the risk of CVD in elderly participants [32
]. This may be attributed to the inclusion of subjects suffering from comorbid diseases; like diabetes and stroke, and other factors that can alter the outcomes viz.
smoking. In addition, the study didn’t clarify the source of the used zeaxanthin; natural or synthetic which has an impact on the bioactivity. Despite the overall result of the study, it pointed out that there were fewer coronary revascularizations in the group receiving lutein and zeaxanthin (2.12%) versus the group receiving placebo (3.27%). Therefore, clinical investigation for the effect of a proper dose of natural zeaxanthin on cardiovascular disorders with the exclusion of any interfering factors for a sufficient period of time is recommended.
Finally, it can be concluded that ZH can be used as a natural therapeutic agent for ameliorating cardiac dysfunction exerting its action through the activation of retinoids receptors in cardiac tissue.