The consumption of Sechium edule (chayote) has antioxidant effect and prevents telomere attrition in older adults with metabolic syndrome

ABSTRACT

Objective

To determine the effect of the consumption of Sechium edule (1.5 g/day) for six months on oxidative stress (OxS) and inflammation markers and its association with telomere length (TL) in older adults with metabolic syndrome (MetS).

Methods

The study was conducted in a sample of 48 older adults: placebo (EP) and experimental (EG) groups. Lipoperoxides, protein carbonylation, 8-OHdG, total oxidant status (TOS), SOD, GPx, H₂O₂ inhibition, total antioxidant status (TAS), inflammatory cytokines (IL6, IL10, TNF-α), and TL were measured before and six months post-treatment.

Results

We found a significant decrease in the levels of lipoperoxides, protein carbonylation, 8-OHdG, TOS in the EG in comparison PG. Likewise, a significante increase of TAS, IL-6, and IL-10 levels was found at six months post-treatment in EG in comparison with PG. TL showed a statistically significant decrease in PG compared to post-treatment EG.

Conclusions

Our findigns showed that the supplementation of Sechium edule has antioxidant, and anti-inflammatory effects, and diminushion of shortening of telomeric DNA in older adults with MetS. This would be the first study that shows that the intervention with Sechium edule has a possible geroprotective effect by preventing telomeres from shortening as usually happens in these patients. Therefore, suggesting a protection of telomeric DNA and genomic DNA.

KEYWORDS:

• Sechium edule
• metabolic syndrome
• telomeric length
• oxidative stress
• antioxidants
• markers inflammatory
• telomerase
• aging

1. Introduction

MetS is characterized by a combination of metabolic abnormalities such as hypertension, atherogenic dyslipidemia, insulin resistance, and abdominal obesity [1]. This syndrome has become a serious global public health problem spread by unhealthy lifestyles [2]. The prevalence of MetS, as well as its components, varies between different ethnicities; mainly because of genetic differences, diet, age, sex, and lifestyles [3]. It is estimated that globally between 20–25% of the adult population, presents MetS [4], and age, is one of the factors that contribute to its prevalence; being the elderly population the most susceptible to present this syndrome [5].

There is scientific evidence to support the fact that people with MetS have a higher risk of cardiovascular events, and the development of neurodegenerative diseases such as Parkinson's, type 2 diabetes mellitus (T2DM), and coronary heart disease, among other disabilities [2,6]. This increases the likelihood of having an acute myocardial infarction or stroke by up to three times compared to people who do not have the syndrome [4]. Even the appearance of MetS at an early age, coupled with a family history of T2DM, predisposes to the development of metabolic diseases in adulthood [7].

MetS is related to the accumulation of oxidized products and deficiency of antioxidant mechanisms, caused by an increase in reactive oxygen species (ROS) [8]. One of the most accepted theories of aging is associated with OxS and it is based on the assumption that cellular lesions or molecular degenerations are due to the accumulation of damage caused by ROS that is simultaneously involved with several age-related pathological conditions, including MetS [9]. Another theory of aging is associated with telomeres, based on the shortening of telomere length (TL) during each cell duplication [10]. Leukocyte telomere length (LTL) is considered a biomarker of cellular aging that also has a close relationship with MetS. Also, is well established that the increase in the number of MetS components is associated with a shortening of the LTL. Likewise, the OxS is also known to be considered a modulator of LTL [11,12].

In this context, it is necessary to find therapeutic strategies that counteract OxS, improve antioxidant protection mechanisms, and avoid or prevent the shortening of LTL and MetS related complications. In this regard, Sechium edule (chayote) is an edible fruit of the Cucurbitaceae family, to which various beneficial properties for human health have been attributed. However, it is still unknown what role it may play in TL dynamics and if it could counteract telomere attrition, and what biochemical, OxS or inflammation markers might be involved. Hence, the objective of this study is to determine the effect of Sechium edule consumption on OxS, inflammatory markers, and its association LTL attrition in older adults with MetS.

2. Materials and methods

2.1. Experimental design

The study was approved on February 23, 2017, by the Research Bioethics and Biosafety Committee of the Faculty of Higher Studies Zaragoza UNAM (23/02-SO/2.4.2) and registered in ISRCTN: 43215432, also the informed and voluntary consent of each of the participants was obtained. It is noted that all procedures were executed following the ethical principles of the Declaration of Helsinki of the World Medical Association. The biological material of the fruit Sechium edule var. nigrum spinosum was donated by the Interdisciplinary Group for the study of Sechium edule of Mexico A.C. (GISeM). The fruits were collected in a state of horticultural maturity; subsequently, they were selected, washed, cut into slices, dried at 40 °C, and pulverized (epidermis, seeds, and spines). The formulation of both active and placebo capsules was carried out in the pharmaceutical development laboratory of the FES-Z. The particle size was standardized to obtain a fine powder of the fruit, with which the capsules were subsequently elaborated based on rheological studies to guarantee the filling, homogeneity, and long-term stability of the powder. Post-design the treatments were manufactured and packaged by a pharmaceutical company specialized in the field of nutraceuticals. The placebo (PG) group received capsules of lactose monohydrate and pharmaceutical talc, both United States Pharmacopeia (USP) grade (Sigma, St. Louis, MO, USA) and with physical characteristics identical to those of the nutraceutical.

For the choice of the dosage of the fruit, an extrapolation of doses between species was carried out through an allometric scaling, based on the normalization of the dose with respect to the surface area and body weight [13]; taking as reference data previously reported by our work group and obtained in an experimental model where different doses of Sechium edule were administered (0, 8, 16, 40,160, 400, 800, 1600, 2900 and 5000 mg/kg) being the LD₅₀ greater than 5000 mg/kg body weight. In this study, a dose starting at 800 mg/kg significantly reduced glucose levels, without altering blood chemistry and without causing toxicity to the liver, kidney, spleen, and thymus [14]. Considering that the dose administered in this model was intraperitoneal with rapid response absorption and total transfer from the peritoneal cavity to the systemic circulation compared to the bioavailability of oral administration [15], a higher dose was selected and intermediate between 800 and 1600 mg/kg, that is, 1200 mg/kg, which, extrapolating in humans, would correspond to 512.64 mg; hence the content was adjusted to 500 mg per capsule. Consecutively, an exploratory study was carried out where the supplementation of the powdered fruit of Sechium edule var. nigrum spinosum was three capsules per day in 12 older adults with MetS in a period of six weeks, evidencing a hepatoprotective, nephroprotective and antioxidant effect after consumption of the fruit [16].

Previously, our working group reported the secondary metabolites present in a capsule of Sechium edule determined by the HPLC technique. These contain in ascending order: 0.71 µg of cucurbitacin I, 6.11 µg of cucurbitacin D, 89.9 µg of cucurbitacin B and 154.8 µg of cucurbitacin E; flavonoids: 0.014 µg of apigenin, 1.3 µg of quercetin, 2.38 µg of myricetin, 14.2 µg of pholirizin, 45.5 µg of rutin and 48.8 µg of naringenin and phenolic acids: 0.11 µg of p-hydroxybenzoic, 1.4 µg of chlorogenic, 1.7 µg of p-coumaric, 3.3 µg of protocatechuic, 7.0 µg of ferulic, 8.7 µg of syringic, 9.3 µg of caffeic and 38.8 µg of gallic [17]. It should also be noted that the antioxidant potential of Sechium edule var. nigrum spinosum was evaluated, both in vivo and in vitro models, using the 2,2-Diphenyl-Picrihydrazil (DPPH) test, a concentration of 1 mg of this fruit can inhibit DPPH by 80%, hence antioxidant effects are attributed to it [18].

The intervention consisted of consuming three capsules (500 mg) each day of Sechium edule or placebo one before each meal, for six months. Convenience sampling was performed in a population of 48 older adults, with an average age of 64.18 ± 4.01. All participants were diagnosed with MetS according to the criteria of the National Adult Treatment Panel of the National Cholesterol Program III (NCEP/ATP III): (i) waist circumference ≥102 cm for men or ≥88 cm for women; (ii) triglycerides ≥150 mg/dL; (iii) high-density lipoprotein cholesterol (HDL-c) < 40 mg/dL in men or <50 mg/dL in women; (iv) blood pressure ≥130/85 mmHg and (v) glucose >110 mg/dL. It was considered a diagnosis of MetS when at least three of the five previous criteria were present [19].

The patients were randomly assigned to the placebo group (PG) (n = 50) or the experimental group (EG) with the intervention of Sechium edule (n = 50). From these patients, only certain participants from the placebo group (n = 23) and the experimental group (n = 25) wished to donate blood for measurements of LTL (Figure 1). All measurements were made before the treatments (baseline measurements) and six months after the intervention.

Figure 1. Flow diagram of the select people.

2.2. Anthropometric measurements

To determine the body dimensions of the patients, weight measurements and waist circumference were performed. For body weight, a calibrated medical scale (SECA, Hamburg, Germany) and for carving a wall stadiometer (SECA, Hamburg, Germany) were used; for this purpose, patients were asked to place their heels together, their heads upright in the Frankfort plane and contact with the stadiometer. The distribution of abdominal fat was obtained by measuring waist circumference with the help of a medical tape measure (SECA, Hamburg, Germany), which was placed at the navel level. All measurements were made by trained personnel from FES-Z [20].

2.3. Blood pressure

To perform clinical measurements such as systolic blood pressure (SBP) and diastolic blood pressure (DBP), a calibrated mercury baumanometer (Medical Rental) was used. The patient was kept at least five minutes at rest before taking blood pressure, sitting with his back straight, feet resting on the ground, and legs without crossing. In addition, the Osler technique was used to identify pseudohypertension [21].

2.4. Biochemical analysis

Blood samples were obtained by venipuncture after a period of 8 h of fasting, which was collected in vacutainer tubes without anticoagulant for determinations of clinical chemistry (glucose, liver function test, lipid and renal profile), telomerase concentration, 8-hydroxydeoxyguanosine (8-OHdG) and serum cytokines. On the other hand, collection tubes with the anticoagulant ethylenediaminetetraacetic acid were used for: glycosylated hemoglobin (HbA1c), H₂O₂ inhibition, and lymphocyte extraction. Finally in tubes with sodium heparin for tests such as lipid peroxidation (LPO), protein carbonylation, superoxide dismutase (SOD), glutathione peroxidase (GPx), total antioxidant status (TAS), and total oxidant state (TOS). Techniques for LPO, protein carbonylation, 8-OHdG, superoxide dismutase (SOD), glutathione peroxidase (GPx), H₂O₂ inhibition, TAS, TOS, and telomerase were performed at the microscale on multiwell plates, which were read in a Multiskan Go, version 1.00.40 (Thermo Scientific, Denver, CO, USA). Glucose, lipid, hepatic and renal profile were determined by colorimetric techniques; while HbA1c by turbidimetric assay by an automated chemical-clinical analyzer Selectra Junior (Vital Scientific, Dieren, Netherlands).

2.5. Thiobarbituric acid reactive substances (TBARS) in plasma samples

The breakdown of unstable peroxides derived from polyunsaturated fatty acids (PUFAs) results in the formation of malondialdehyde (MDA), which can be colorimetrically quantified after reaction with thiobarbituric acid (TBA) in the presence of phosphoric acid (H₃PO₄); giving rise to the formation of the TBA-MDA adduct, evidenced by a pink hue with absorption at 535 nm. To avoid a possible amplification of lipid peroxidation; the antioxidant butylhydroxytoluene (BHT) was added [22]. To do this, 200 μL of plasma, 25 μL of BHT (12.6 mM) (Sigma, St. Louis, MO, USA), 200 μL of H₃PO₄ (0.2 mol/L) (Sigma, St. Louis, MO, USA), and 25 μL of TBA (0.11 mol/L) (Sigma, St. Louis, MO, USA) were incubated for 45 min at 90°C. To stop the reaction, the mixture was cooled in ice and 500 μL of butanol (Sigma, St. Louis, MO, USA) was added, along with 50 μL of saturated sodium chloride (NaCl) solution (Sigma, St. Louis, MO, USA). The absorbance was read at a wavelength of 535 and 572 nm, the latter to correct the reference absorption. Finally, the quantification was carried out using a calibration curve.

2.6. Carbonylated proteins

The carbonylated proteins were determined by the 2,4-dinitrophenylhydrazine (DNPH) assay, which is based on the reaction between carbonyls with DNPH forming phenylhydrazones which precipitate giving a yellow-orange coloration. 20 μL of DNPH (10 mM) (Sigma, St. Louis, MO, USA) were mixed into H₃PO₄ (0.5 M) (Sigma, St. Louis, MO, USA) and 20 μL of plasma; this mixture was incubated for 10 min at room temperature with constant stirring. Subsequently, 20 μL of sodium hydroxide (NaOH) (6 M) was added (Sigma, St. Louis, MO, USA). Dinitrophenyldidrazines are highly unstable in an alkaline medium; therefore, the incubation period after the addition of NaOH was rigorously monitored [23]. The absorbance was measured at a wavelength of 450 nm. The total protein concentration was determined using the Bradford reagent (Bio-Rad, Hercules, CA, USA) and a bovine serum albumin (BSA) standard (1. 41 mg/mL). The samples were read at a wavelength of 595 nm and the results were expressed as mg/mL of protein.

2.7. DNA oxidative damage

For the quantification of the 8-OHdG molecule, the commercial kit (Wuhan Fine Biotech Co. Ltd., Hubei, China) was used, based on the competitive ELISA detection method in pre-incubated plates. During the reaction, the presence of the antigen in the samples was determined by the ability to compete with the fixed reference antigen on the plate by binding to the antibody. The reaction was terminated by adding sulfuric acid (H₂SO₄) and the color change was measured spectrophotometrically at 450 nm. The concentration of 8-OHdG was determined by interpolating the optical density (O.D.) of the samples on a standard curve.

2.8. SOD enzymatic activity

The activity of the SOD enzyme was determined by the commercial kit (Randox Laboratories Ltd., Antrim, UK), following the manufacturer's instructions. Xanthine oxidase was used as a generator of superoxide radicals, which in turn react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyl-tetrazolium chloride to form the formazan dye, read at a wavelength at 505 nm. The inhibition of this reaction, when SOD was present, allowed to determine its activity.

2.9. GPx enzymatic activity

The activity of the GPx enzyme was determined spectrophotometrically by the commercial kit (Randox Laboratories Ltd., Antrim, UK). Based on the principle that GPx catalyzes the oxidation of reduced glutathione (GSH) by cumene hydroperoxide then oxidized glutathione (GSSG) in the presence of glutathione reductase and nicotinamide adenine dinucleotide reduced phosphate (NADPH) is immediately converted into its reduced form, while NADPH passes into its oxidized form to nicotinamide adenine dinucleotide phosphate (NADP+). The decrease in absorbance was measured at 340 nm.

2.10. H₂o₂ inhibition

The H₂O₂ inhibition was determined by spectrophotometry, using hydrogen peroxide (H₂O₂) as a substrate (Sigma, St. Louis, MO, USA). A mixture of 190 μL of working solution (phosphate buffer 0.1 M, pH = 7.0 and H₂O₂ 20 mM) and 10 μL of the sample was made. The decrease in H₂O₂ concentration was measured every 15 s for 2 min at a wavelength of 240 nm [24].

2.11. Total antioxidant status (TAS)

The total antioxidant status was determined by the commercial kit (Randox Laboratories Ltd., Antrim, UK); based on the reaction between 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) with metmyoglobin and H₂O₂ which stimulates the formation of the cationic radical ABTS⁺; a characteristic of this radical is its blue–green coloration. So the intensity of the hue was inversely proportional to the amount of antioxidants present in the sample. The kinetics of the reaction was determined at a wavelength of 600 nm.

2.12. Total oxidant status (TOS)

Plasma TOS determination was performed using the commercial kit (Rel Assay Diagnostics, Gaziantep, TR), following the supplier's instructions. This test has a principle that the oxidants present in the sample can oxidize the ferrous ion (Fe²⁺)-chelator complex to ferric ion (Fe³⁺). Fe³⁺ forms a complex colored with chromogen in an acidic medium. So the intensity of the color (which was measured spectrophotometrically at a wavelength of 530 nm) was directly related to the amount of oxidizing molecules present in the simple.

2.13. Inflammatory cytokines

The levels of interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-α) were determined in serum samples by the cytokine bead array (CBA) Human assay; using the human inflammatory cytokine kit (BD Biosciences, San Jose, CA, USA), which consists of a sandwich capture method with beads conjugated with specific antibodies and a detection reagent (phycoerythrin (PE) mixture)-conjugated antibodies), which results in a fluorescent signal in proportion to the number of bound analytes, which were detected by the flow cytometer (BD Biosciences, San Jose, CA, USA), and the FCAP ArrayTM version 3.0 software.

2.14. Serum telomerase levels

The quantitative analysis of the enzyme telomerase (TE) was determined in serum samples by a sandwich-type ELISA immunoassay (MyBioSource, San Diego, CA, USA) where the antigen present in the sample was immobilized between two antibodies, one capture and one detection conjugated to the enzyme horseradish peroxidase (HRP), a substrate is added that when reacting with the enzyme provides a visible signal that allows the detection and quantification of TE at 450 nm. The concentration of the enzyme telomerase was proportional to the value of the O.D.

2.15. Lymphocyte isolation and DNA extraction

Lymphocyte isolation was performed from 5 mL of 1:1 diluted whole blood with saline phosphate buffer (PBS) (Sigma, St. Louis, MO, USA)/fetal bovine serum (FBS) at 2% (ThermoFisher Scientific, Waltham, MA, USA); subsequently, 4 mL of Ficoll-paque (Gibco ThermoFisher Scientific, Waltham, MA, USA) was added. The opaque interface of interest was centrifuged and separated; the samples were stored in cryovials with a freezing mixture (Dimethyl sulfoxide (DMSO)) at 5% (Sigma, St. Louis, MO, USA), FBS 20% and a half of Iscove modified Dulbecco (IMDM) (Gibco ThermoFisher Scientific, Waltham, MA, USA) at −80°C until use. Finally, from 2 × 10⁶ lymphocytes, DNA extraction was performed, using the isolation kit (Qiagen, Hilden, Düssseldorf, Germany), following the manufacturer's recommendations. The DNA extraction was performed just before the determination of the TL, and its quality and integrity were subsequently determined.

2.16. Leukocyte telomere length (LTL)

From 5 ng of genomic DNA, the average length of telomeres in plates of 96 wells (Applied Biosystems, Waltham, MA, USA) was determined by quantitative polymerase chain reaction (qPCR) with the 7500 Real-Time PCR equipment (Applied Biosystems, Waltham, MA, USA). Using the Absolute Human Telomere length quantification qPCR Assay Kit (AHTLQ) (Sciencell, Carlsbad, CA, USA). In each of the samples analyzed, two reactions were carried out: one that amplified the single-copy reference gene (SCR) and another for telomere sequencing. SCR primers recognize and amplify a region 100 bp in length of human chromosome 17; therefore, it serves as a reference for data normalization. Both reactions were taken to a final volume of 20 μL (1 μL reference genomic DNA sample or 5 ng genomic DNA template, 2 μL of first stock solution (telomere or SCR), 10 μL 2x qPCR master mix FastStart Essential DNA Green Master (Roche Diagnostics GmBH, Mannheim, Germany) and about 7 μL of nuclease-free H₂O. The PCR conditions were as follows: 1 cycle of initial denaturation, 95 °C for 10 min and 32 cycles of denaturation, 95 °C for 20 s; annealing, 52 °C by 20 s; extension, 72 °C for 45. For the analysis of results, the comparative ΔΔCq (Quantification Cycle Value) method was used.

2.17. Statistical analysis

The results are shown as mean ± standard deviations; data were analyzed by ANOVA of repeated measures. The associations between LTL and the different analyzed parameters were determined using the Pearson correlation coefficient, using the IBM SPSS V 20 statistical program (Armonk, NY, USA). Results with statistical significance were considered when p < 0.05. All determinations were made in duplicate.

3. Results

Table 1 shows clinical and anthropometric measurements by study group before and after treatment (six months). Regarding anthropometric measurements, the EG presented a decrease with borderline statistical significance in terms of body weight at six months post-treatment compared to the PG. Likewise, the SBP and DBP clinical measurements also showed a statistically significant decrease at six months post-treatment in the EG.

Table 1. Clinical and anthropometric measurements by study group.

Parameter	Placebo Group n = 23	Experimental Group n = 25	p-value
Body weight (kg)
Baseline	81.6 ± 21	78.1 ± 16
6 months	81.5 ± 21	76.2 ± 17*	0.05
Waist circumference (cm)
Baseline	106 ± 16	105 ± 11
6 months	105 ± 17	104 ± 13	0.08
SBP (mmHg)
Baseline	132 ± 9	133 ± 14
6 months	127 ± 16	126 ± 13*	0.02
DBP (mmHg)
Baseline	88.9 ± 9	84.6 ± 10
6 months	88.4 ± 11	80.0 ± 10*	0.01

The data are expressed as the average ± standard deviation. ANOVA of repeated measures, significance level 95%, p < 0.05. SBP, systolic blood pressure; BPD: diastolic arterial pressure. *Baseline statistical significance vs 6 months inter-group.

Regarding the biochemical parameters, the EG showed a statistically significant decreasing urea and HbA1c% levels and a statistically significant increase in HDL-c concentration at six months post-treatment compared to PG. On the other hand, total cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, direct bilirubin, uric acid, and creatinine, did not present statistically significant differences (Table 2).

Table 2. Pre and post-treatment biochemical parameters by study group.

Parameter	Placebo Group n = 23	Experimental Group n = 25	p-value
Glucose [mg/dL]
Baseline	118 ± 67	117 ± 73
6 months	123 ± 48	108 ± 52	0.21
Cholesterol [mg/dL]
Baseline	195 ± 35	201 ± 51
6 months	182 ± 34	207 ± 40	0.29
HDL-c [mg/dL]
Baseline	47.5 ± 8	44.5 ± 7
6 months	7.3 ± 8	48.2 ± 9*	0.03
Triglycerides [mg/dL]
Baseline	187 ± 58	181 ± 76
6 months	161 ± 53	169 ± 74	0.08
AST [U/L]
Baseline	28.0 ± 8	28.3 ± 10
6 months	24.7 ± 7	25.8 ± 7	0.36
ALT [U/dL]
Baseline	28.4 ± 10	34.1 ± 17
6 months	27.0 ± 11	26.1 ± 12	0.71
Total bilirubin [mg/dL]
Baseline	0.58 ± 0.2	0.56 ± 0.2
6 months	0.53 ± 0.2	0.54 ± 0.2	0.68
Direct bilirubin [mg/dL]
Baseline	0.21 ± 0.10	0.20 ± 0.08
6 months	0.18 ± 0.07	0.23 ± 0.08	0.11
Uric acid [mg/dL]
Baseline	5.0 ± 1.5	4.8 ± 1.3
6 months	4.6 ± 1.2	5.1 ± 1.4	0.69
Urea [mg/dL]
Baseline	34.8 ± 10	37.6 ± 11
6 months	34.3 ± 10	31.9 ± 8*	0.05
Creatinine [mg/dL]
Baseline	0.85 ± 0.2	0.89 ± 0.1
6 months	0.81 ± 0.1	0.82 ± 0.1	0.41
HbA1c (%)
Baseline	6.98 ± 1.6	7.20 ± 1.2
6 months	6.94 ± 2.0	6.28 ± 1.2*	0.01

The data are expressed as the mean ± standard deviation. ANOVA of repeated measures, significance level 95%, p < 0.05. HDL-c: high-density lipoprotein cholesterol; AST, aspartate aminotransferase; ALT, alanine aminotransferase; HbA1c: glycosylated hemoglobin. *Baseline statistical significance vs 6 months inter-group.

Markers of OxS showed a statistically significant decrease in the levels of lipoperoxides, protein carbonylation, and DNA damage, at six months post-treatment in EG compared to PG. On the other hand, the activity of SOD, H₂O₂ inhibition, and total antioxidant capacity (TAS) showed an increase in the EG. Likewise, the TOS showed a statistically significant decrease in the EG compared to the PG (Table 3).

Table 3. OxS markers and antioxidant capacity by study group.

Parameter	Placebo Group n = 23	Experimental Group n = 25	p-value
Lipoperoxides [µmol/L]
Baseline	0.23 ± 0.05	0.29 ± 0.11
6 months	0.21 ± 0.02	0.19 ± 0.03*	0.01
Protein carbonylation [nmol/mg]
Baseline	30.3 ± 5.3	31.6 ± 5.3
6 months	34.6 ± 6.9	28.7 ± 4.6*	0.05
8-OHdG [ng/mL]
Baseline	30.3 ± 8.0	32.0 ± 9.3
6 months	31.1 ± 8.5	21.5 ± 7.4*	0.01
SOD [U/mL]
Baseline	177 ± 13	181 ± 7
6 months	163 ± 14	185 ± 6*	0.05
GPx [U/L]
Baseline	5007 ± 2091	6611 ± 1792
6 months	4315 ± 1362	6726 ± 2864	0.08
H2O2 Inhibition [µmol/min]
Baseline	3.26 ± 1.1	3.14 ± 1.19
6 months	2.42 ± 0.5	3.77 ± 1.03*	0.01
TAS [mmol/L]
Baseline	1.33 ± 0.19	1.06 ± 0.19
6 months	1.05 ± 0.25	1.26 ± 0.25*	0.001
TOS [µmol H2O2 Equiv./L]
Baseline	24.9 ± 2.2	29.1 ± 4.3
6 months	28.5 ± 4.2	24.7 ± 4.9*	0.05

The data are expressed as the mean ± standard deviation. ANOVA of repeated measures, significance level 95%, p < 0.05. 8-OHdG: 8-hydroxydeoxyguanosine; SOD, superoxide dismutase; GPx, glutathione peroxidase; TAS: total antioxidant status. TOS: total oxidizing state. *Baseline statistical significance vs 6 months inter-group.

In the measurement of markers related to the chronic inflammatory process, a statistically significant increase in IL-6 and IL-10 levels was found at six months post-treatment in EG in comparison with PG, while TNF-α levels showed no significant changes (Table 4).

Table 4. Pre and post-treatment inflammatory markers by study group.

Parameter	Placebo Group n = 23	Experimental Group n = 25	p-value
IL-6 [pg/dL]			0.01
Baseline	8.17 ± 4.6	7.5 ± 5.8
6 months	8.82 ± 1.6	11.4 ± 3.5*
IL-10 [pg/dL]			0.01
Baseline	3.83 ± 1.3	2.48 ± 1.3
6 months	3.05 ± 0.5	4.15 ± 1.0*
TNF-α [pg/dL]			0.22
Baseline	5.11 ± 1.9	4.34 ± 2.0
6 months	4.34 ± 1.3	4.90 ± 1.4

Data are expressed as means ± standard deviation. ANOVA of repeated measures test, significance level 95%, p < 0.05. IL-6: interleukin-6; IL-10: interleukin-10; TNF-α: tumor necrosis factor-alpha. *Baseline statistical significance vs 6 months inter-group.

Telomerase levels showed a statistically significant increase in PG compared to post-treatment EG. Likewise, LTL showed a statistically significant decrease in PG compared to post-treatment EG (Table 5).

Table 5. Pre and post-treatment telomerase levels and telomere length by study group.

Parameter	Placebo Group n = 23	Experimental Group n = 25	p-value
Telomerase [ng/mL]			0.02
Baseline	3.45 ± 0.8	4.66 ± 1.4
6 months	4.23 ± 0.7*	4.40 ± 1.1
Telomere length [pb]			0.01
Baseline	20.2 ± 4.2	19.4 ± 6.0
6 months	14.5 ± 4.2*	25.3 ± 7.8
Delta	−5.6 ± 0.18	6.33 ± 1.24

Data are expressed as means ± standard deviation. ANOVA of repeated measures test, significance level 95%, p < 0.05. *Baseline statistical significance vs 6 months inter-group.

Table 6 shows the parameters that presented a statistically significant correlation with respect to TL. In this sense, a positive association was found between H₂O₂ inhibition, and IL-6 levels, coupled with a negative association between protein carbonylation and 8-OHdG with respect to LTL in EG after 6 months post-treatment.

Table 6. Correlation between OxS, and inflammation with respect to LTL pre and post-treatment by study group.

Group/marker	LTL	Carbonylation	8-OHdG	H2O2 inhibition	IL-6
PG	Baseline r= p =	−0.120 0.696	−0.383 0.219	0.060 0.846	−0.061 0.835
	6 months r= p =	−0.053 0.857	−0.426 0.191	0.073 0.813	−0.038 0.898
EG	Baseline r= p =	−0.434 0.390	−0.410 0.189	0.034 0.872	−0.045 0.200
	6 months r= p =	−0.789* 0.001	−0.563* 0.036	0.737* 0.001	0.503* 0.010

The data express the bivariate correlations between the different parameters analyzed with respect to leukocyte telomere length (LTL). r = Pearson correlation coefficient, p ≤ 0.05. HDL-c: high-density lipoprotein cholesterol; 8-OHdG: 8-hydroxydeoxyguanosine; H₂O₂ inhibition; IL-6: interleukin-6; PG, placebo group; GE: experimental group.

4. Discussion

One of the characteristics that frequently occur in patients with MetS is abdominal obesity which can be considered an indicator of dysfunctional adipose tissue that does not adequately store excess energy [25]. In this sense, Sechium edule has been attributed anti-obesity properties [26]; which was evidenced in the present study, given that a decrease in body weight (by about two kilograms) could be observed in patients who consumed Sechium edule for six months compared to those who only received placebo. This coincides with a previous study, where MetS patients who consumed 1500 mg of Sechium edule for three months, had a decrease in body weight of about one kilogram [27]; therefore, its effect lasts and is accentuated in the long term. This decrease can be attributed to the low caloric and high fiber content contained in chayote [28,29] and/or its ability as an inhibitor of lipogenesis (fat synthesis from dietary carbohydrates) or a lipolysis stimulator (fat breakdown) via AMPK activation (AMP-activating protein kinase) [30].

On the other hand, high blood pressure is a serious medical condition that significantly increases the risk of brain, kidney, and heart disease. An estimated 1.3 billion people worldwide have high blood pressure, but only 18% have it under control [31]. Sechium edule has been reported to exert a hypotensive effect on pre-diabetic and hypertensive individuals [32,33]. This was corroborated in our study, SBP and DBP decreased by 7 and 4 mmHg respectively in EG at six months of treatment. The hypotensive effect may be caused by the presence of polyphenolic compounds in Sechium edule, such as quercetin and coumaric acid that provide vasodilator actions. While the possible mechanism of action would be on the AT1 receptors of the vasoconstrictor angiotensin II and/or calcium flows activated by AGII [34].

In experimental and human models, the hypoglycemic effect of Sechium edule has been confirmed [35,36]; this was validated in the present study since the EG showed a statistically significant decrease in the percentage of HbA1c, which has been associated with a reduction of the complications of T2DM. This may be due to the presence of bioactive compounds such as flavonoids in chayote, which act through the inhibition of the enzymes α-amylase and α-glucosidase, delaying the absorption and digestion of carbohydrates from the digestive tract, favoring the reduction of blood glucose [37,38]. It has been shown that the flavonoids present in the fruit, such as quercetin and naringenin improve HDL-c functions through their antioxidant effects, which may have occurred in post-treatment EG, as HDL-c levels also increased when supplemented with the fruit. Hence, Sechium edule has been attributed a cardioprotective activity [16,39,40].

Another beneficial effect of Sechium edule is to perform a protective effect against nephrotoxicity and restoration of renal function; since in diabetic experimental models treated with aqueous extracts of Sechium edule at a concentration of 200 mg/kg of body weight, an improvement in kidney histology related to a decrease in blood urea (main residue of protein breakdown) was observed [41]. The latter coincides with what was observed in the present study since the EG showed a decrease in urea levels by about 7% at six months post-treatment compared to the PG. So, the consumption of the fruit is not nephrotoxic and could even have a nephroprotective effect.

Regarding the results obtained on oxidative damage, a reduction was found in the markers of OxS at different levels (lipids, proteins, and DNA). It is important to note that lipoperoxides in people who consumed the fruit decreased by almost 10% in EG in comparison with PG. In this sense, in a previous study, where the intervention with Sechium edule was during a period of three months, the reduction was 5% [27]. This indicates that the reduction of OxS damage at the lipid level persists and is accentuated with the consumption of the fruit, one of the possible mechanisms being the inhibitory capacity exerted by flavonoids such as quercetin and rutin; attributable to its chelating and free radical scavenging activities dependent on iron ions, which form inert complexes, unable to initiate the chain reaction and extension of lipid damage [42]. Likewise, the chelating activity of flavonoids has been shown to reduce oxidative damage in proteins [43]. In addition to the above, there is scientific evidence that supports the correlation between the aging process and various pathologies with the increase in protein oxidation, which can cause non-reversible modifications causing proteins to lose their functionality [44]. Also, oxidative damage at the protein level was evidenced by the statistically significant decrease in carbonylated proteins in the EG; a constant in MetS patients [45]. It should be noted that protein carbonylation was counteracted by almost 17% by the intervention of fruit consumption for six months; even supplementation for shorter periods has the same antioxidant effect [27].

DNA damage by OxS can lead to inactivation or loss of genes and if this damage is not repaired it can accumulate and be passed on to subsequent cell generations; leading to mutations that can lead to death [46]. In our study, the levels of oxidative damage at the DNA level were mitigated by 32% in the EG after consumption of the fruit; this effect can be attributed to the antioxidant capacity of naringenin, since this compound decreases oxidative damage levels at the DNA level in renal and hepatic tissues, attenuating nephrotoxicity and hepatotoxicity, or also to the antioxidant capacity exerted by quercetin and apigenin, flavonoids that can enter the cell nucleus and suppress oxidative damage [47–49]. Therefore, the consumption of the fruit could have beneficial effects, since the decrease of the biomarker 8-OHdG can avoid the promotion of carcinogenesis. Also, it has been proven the antiproliferative capacity exerted by different varietals of chayote against cell lines including HeLa or leukemia of mouse macrophages P388 is proven [50].

Endogenous antioxidants are defense mechanisms that have the ability to neutralize ROS by preventing them from becoming toxic compounds. In this sense, the enzymatic activity of SOD and H₂O₂ inhibition showed a statistical decrease in PG; in contrast to EG, where increased at six months post-treatment. The increase in the activities of these enzymes may be due to the presence of quercetin, which enhances the binding of the master regulator of the antioxidant response, known as erythroid-derived nuclear factor 2 (Nrf2) to DNA [51,52]. Likewise, the increase in the activities of enzymes with antioxidant function may be associated with the decrease of oxidative damage at the level of lipids, proteins, DNA, and TOS and an increase of TAS in the EG via Nrf2. TAS refers to the increased bioavailability and ability of antioxidant compounds present in the system to reduce oxidizing species. Therefore, increased levels of TAS in EG could mean better antioxidant defense capacity to counteract OxS in MetS patients; supporting the proposal that chayote has a concentration of antioxidants capable of exerting a preventive effect for chronic non-communicable diseases [53]. The above may be related to the neutralization of the TOS which refers to the total amount of oxidant molecules present in the system, in this sense a statistically significant decrease of about 13% was observed in the EG. Therefore, the increase in TAS and the decrease in TOS may suggest the presence of better mechanisms to mitigate oxidative stress in the EG [54]. It should be noted that most of the information reported so far on the bioactive compounds present in the fruit such as cucurbitacins, flavonoids, and phenolic acids, and their association with TL and telomerase activity has been studied in cancer cells. Hence the need to delve into the possible protective effects of these compounds on TL in human cells without carcinogenic processes. However, there is a large body of evidence on the ability of these compounds to eliminate ROS.

Figure 2 shows the possible mechanism of Sechium edule to avoid shortening telomere length in patients with MetS.

Figure 2. Proposal of the possible mechanism of Sechium edule to avoid shortening telomere length in patients with MetS.

Regarding cucurbitacins, it has been reported that they present antioxidant and chemoprotective activity associated with a decrease in oxidant damage at the level of lipids, proteins, and DNA; as well as the inflammatory process in both in vivo and in vitro models [55–57]. In the case of cucurbitacins I, D, B, and E, they promote the expression of phase II detoxification enzymes heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO-1) through modulation of the Nrf2 transcription factor, which in turn regulates the expression of genes whose protein products participate in antioxidant protection mechanisms (SOD, GPx, and CAT). Likewise, these cucurbitacins cause the inhibition of nuclear factor enhancing the kappa light chain of activated B cells (NFkB) (transcription factor related to the inflammatory process) [58].

Flavonoids such as apigenin promote the expression of SOD, GPx, and CAT with the consequent decrease in lipid oxidation; coupled with the expression of Nrf2 in vitiligo perilesional melanocyte cells [59]. Quercetin also protects against oxidative damage, through redox modulation of the Nrf2-dependent glutathione system [60]. While myricetin increases the nuclear accumulation of Nrf2 and inhibits NFkB, generating a protective effect on diabetic cardiomyopathy [61,62]. For its part, phlorizin stimulates the translocation of Nrf2 from the cytoplasm to the nucleus and upregulates its downstream antioxidant response element (ARE), which includes the enzymes HO-1 and NQO-1 and inhibits apoptosis in an animal model with oxidative injury induced by exhaustive exercise [63]. For its part, naringenin reduces ROS levels and improves mitochondrial dysfunction through the Nrf2/ARE pathway in neurons from hypoxia-treated rats [64].

About the phenolic acids present in Sechium edule, they have been reported to have antioxidant and anti-inflammatory effects mediated by Nrf2 and NFkB, respectively. In the case of chlorogenic acid, it exerts a nephroprotective effect by inhibiting OxS and inflammation [65]. The p-coumaric acid and the protocatechuic have antioxidant effects [66], in addition, the p-coumaric has hepatoprotective [67] and neuroprotective effects due to the inhibition of the NFkB p65 subunit [68]. Likewise, it has been pointed out that ferulic acid mitigates inflammatory lesions and apoptosis caused by cadmium chloride and maintains the redox balance in testicular tissue [69]. Syringic acid decreases testicular oxidative damage caused by treatment with methyl cellosolve at the lipid level, associated with an increase in antioxidant enzymes such as SOD, GPX, and CAT [70]. For its part, caffeic acid prevents hepatotoxicity caused by acetaminophen, increasing the expression of HO-1 and NQO-1 [71], it also exerts anti-inflammatory actions, due to the uptake of nitric oxide (NO) and its capacity to modulate the expression of inducible nitric oxide synthase (iNOS) [72]. And finally, gallic acid increases nuclear Nrf2 levels and attenuates oxidative damage [73].

With respect the anti-inflammatory effect of chayote, we found that the markers IL-6 and IL-10 increased in post-treatment EG, which coincides with what was previously reported by our working group [17]. The role of IL-6 in immunity depends on the local concentration, as well as the presence or absence of regulatory proteins acting in the signal transduction pathway or the concentration of its soluble receptor [74]. Loss of IL-6 signaling has been shown to cause increased susceptibility to OxS and cell death; hence, its increase influences the antioxidant response mediated by Nrf2 and survival; this is achieved by decreasing mitochondrial activity and stimulating selective mitophagy; which leads to mitigate ROS levels and therefore greater survival of pancreatic β cells in diabetogenic conditions [75]. It is also recognized that IL-6 is mediated via STAT downstream, which decreases OxS by upregulation of SOD [76]. This coincides with the results observed in the EG at six months of treatment since there was an increase in the levels of SOD and IL-6. One of the bioactive components of chayote to which the anti-inflammatory effect can be attributed is naringenin, as it has been shown to counteract cell death of β cells, inhibiting both the extrinsic and intrinsic pathway of apoptosis, in addition to these protective effects being associated with the suppression of oxidative damage to DNA, through the signaling pathways mediated by NFκB, together with that of mitogen-activated protein kinase [77]. IL-10, on the other hand, is an anti-inflammatory interleukin that controls the duration and degree of inflammation capable of inhibiting the synthesis of pro-inflammatory cytokines via STAT3 [78]. In this regard, it has been shown that low levels of IL-10 condition the development of tissue lesions and morphological damage [79,80], while mutations at the gene level, resulting in increased production of pro-inflammatory cytokines predisposing to increased risk of developing T2DM [81]. The increase in IL-10 levels in the EG may be due to the presence of quercetin and luteolin in chayote since it has been shown that low concentrations of these bioactive compounds are able to stimulate the expression of this anti-inflammatory cytokine [82].

It has been established that the shortening of the TL can predict the deterioration of the metabolic condition of the individual, which predisposes to the development of various diseases [83]. In this sense, it has been identified that individual MetS alterations by themselves shorten the length of telomeres; while the sum of them can act synergistically and further enhance wear [84,85]; however, it should be noted that dyslipidemia is an exception, since the reported results are contradictory [86,87]. Most of the current studies have been carried out in people with MetS compared to healthy people, but with age ranges ranging from 18–75 years; very few have followed up [83,88,89]. This would be the first study conducted exclusively in older adults with MetS treated with Sechium edule (as a possible reducer of the rate of TL wear) and followed up for six months. The presence of MetS in the PG caused a 28% shortening of the LTL compared to that observed in the EG. The shortening observed in the PG may be due to the persistence of oxidative damage at different levels (lipids, proteins, and DNA) or due to the increase in the content and/or reactivation of telomerase; the latter leads to a state of constant inflammation due to a delay in apoptosis, which causes tissue damage and disease progression, thus contributing to the pathogenesis of atherosclerosis in these patients [90,91]. As can be seen in the group that received the intervention, there was a tendency to increase LTL. Therefore, correlations and analysis of covariance (data not shown) were performed, adjusting the delta of length to that of weight. Where it was possible to show that the tendency to increase LTL is due to treatment and not to weight loss in patients with MetS.

As previously mentioned, Sechium edule supplementation prevents the shortening of the TL in the EG, which we assume to be a consequence of the decrease in oxidative damage, mainly at the DNA level. Based on the results, we can propose that the bioactive compounds present in the fruit act as OxS antagonists and possibly prevent telomere shortening in people with MetS. One of the biggest difficulties in the treatment of this syndrome lies in the use of drugs together for long periods with the possibility of side effects. Hence, new interventions have helped to respond promptly to the control or prevention of the different MetS conditions; as well as telomeric wear [84].

In this sense, this would be the first study that shows that the intervention with Sechium edule has a possible geroprotective effect by preventing telomeres from shortening as usually happens in these patients. This effect would be directly associated with the reduction of oxidative damage at the DNA level as a result of the increase in the mechanisms of antioxidant protection and inhibition of the inflammatory process mediated by Nrf2 and NFkB, respectively. Telomerase content and activity have been shown to be increased in people with MetS [27,90]. In the present study, the above was confirmed, together with the shortening of the LTL in the GP. Therefore, overexpression and/or increased activity of this enzyme do not prevent wasting [92]. In contrast, in the EG both the enzyme levels and the LTL did not present changes after treatment. Therefore, it is proposed that the maintenance of LTL in the EG is independent of telomerase.

Based on our results, it can be proposed that LTL shortening is related to an increase in telomerase levels in patients with MetS of the PG; probably to recover and maintain TL; although this was not effective enough to counteract the permanent damage caused by OxS at in proteins, lipids, and DNA. This can have a profound impact in such a way that it does not only affect the patient with MetS but also their offspring; which predisposes them to develop chronic non-communicable diseases. The decrease in LTL and the increase in telomerase content in the PG are consistent with those reported in autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, also in children who suffered burns and individuals with chronic obstructive pulmonary disease (COPD) [93–95]. In patients with immune thrombocytopenia [96], atopic dermatitis, psoriasis [97], parthenium-induced contact dermatitis [98], in mycosis fungoides and parapsoriasis this pattern may reflect tumorigenesis [99].

It has also been reported that, in some cases, the increase in telomerase expression is associated with the development of cancer; hence the fact of highlighting the link between the different components of MetS such as dyslipidemia, T2DM, obesity, and inflammation with a greater predisposition to the development of breast cancer, colorectal cancer, pancreas, among others [100,101].

Meanwhile, in EG telomerase levels and LTL remained stable; therefore, our findings suggest that consumption of Sechium edule leads to greater stability of genomic DNA [102], which could prevent the development of carcinogenic processes due to its antiproliferative effect [50]. In this regard, the decrease in OxS at the level of lipids, proteins, and DNA, can have a beneficial impact on the health of these individuals, counteracting the complications of the components of MetS.

Finally, significant associations were observed between some molecular parameters with LTL, especially in the case of protein carbonylation and H₂O₂ inhibition, in which a negative and positive association was found respectively. These findings suggest that LTL is favored if oxidative damage at the protein level decreases or if H₂O₂ inhibition is increased, the latter coincides with a study conducted on expert athletes, compared to untrained people [103].

On the other hand, the mean negative association between the 8-OHdG molecule and the LTL can be attributed to the consumption of Sechium edule; this finding suggests that TL may be favored if levels of oxidative DNA damage decrease. In this sense, healthy lifestyles such as adherence to a Mediterranean diet (lower consumption of carbohydrates and meats and a higher proportion of nuts, vegetables, and fiber) are positively related to LTL [104]. Likewise, a positive association between IL-6 with respect to LTL was observed at six months post-treatment in EG. This finding coincides with what was previously reported by our working group where fat mass decreased in patients who consumed Sechium edule [17]. In fact, intracerebroventricular administration of IL-6 has been shown to reduce body fat in experimental models [74]. Finally, it is important to point out the limitations of the present study, one of them is to demonstrate the power of the fruit by comparing it with some other phytochemical, such as quercetin, the non-representative sample size as well the fact that the groups were also not proportional by sex, so the influence of this variable could not be evaluated. However, our analysis of the variance of repeated measures allows us to show changes in differences between groups, even if the value of the quantified parameter is different from the reference value. In this sense, it would be advisable to conduct a longitudinal study with further clinical evaluations and a cross-over design along with representative sample size. To confirm our findings, it is necessary to carry out the measurement of telomere attrition by Terminal Restriction Fragment (TRF) analysis, because this is considered the ‘gold standard’ technique.

5. Conclusions

Our findings show that Sechium edule has anti-obesity, hypotensive, hypoglycemic, antioxidant, anti-inflammatory, and geroprotective effects that may influence the maintenance of telomerase levels and LTL in people with MetS, the latter associated with a decrease in OxS damage, leading to greater stability of genomic DNA. Therefore, the consumption of this fruit can be proposed as a complementary therapeutic alternative to prevent or counteract the appearance of the different components of MetS and unwanted immune responses; among other diseases related to the aging process.

Author contributions

V.M.M.-N designed the study, wrote the manuscript, and analyzed the data. J.R-P., T.L.A.-U., I.A.-S., E.S.-O. and G.G-G. performed the study and analyzed the data. All authors reviewed the final manuscript.

Institutional review board statement

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of the "National Autonomous University of Mexico (UNAM) - Zaragoza Campus"’.

Informed consent statement

Informed consent was obtained from all subjects involved in the study.

Acknowledgments

We appreciate the support from National Council for Science and Technology (CONACyT) for the scholarship granted to Graciela Gavia-García for postdoctoral stay.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are available from the corresponding author, [VMM-N], upon reasonable request.

Additional information

Funding

This work was supported by grants from the General Directorate of Academic Personnel Affairs, National Autonomous University of Mexico (DGAPA-UNAM) (PAPIIT IN215821), and the Secretariat of Science and Technology and Innovation Project of Mexico City (SECITI) (SECITI/045/2018).