Spondias mombin supplementation attenuated cardiac remodelling process induced by tobacco smoke

Abstract The objective of this study was to investigate the influence of Spondias mombin (SM) supplementation on the cardiac remodelling process induced by exposure to tobacco smoke (ETS) in rats. Male Wistar rats were divided into 4 groups: group C (control, n = 20) comprised animals not exposed to cigarette smoke and received standard chow; group ETS (n = 20) comprised animals exposed to cigarette smoke and received standard chow; group ETS100 (n = 20) received standard chow supplemented with 100 mg/kg body weight/d of SM; and group ETS250 (n = 20) received standard chow supplemented with 250 mg/kg body weight/d of SM. The observation period was 2 months. The ETS animals had higher values of left cardiac chamber diameters and of left ventricular mass index. SM supplementation attenuated these changes. In addition, the myocyte cross‐sectional area (CSA) was lower in group C compared with the ETS groups; however, the ETS250 group had lower values of CSA compared with the ETS group. The ETS group also showed higher cardiac levels of lipid hydroperoxide (LH) compared with group C; and, groups ETS100 and ETS250 had lower concentrations of LH compared with the ETS group. Regarding energy metabolism, SM supplementation decreased glycolysis and increased the β‐oxidation and the oxidative phosphorylation. There were no differences in the expression of Nrf‐2, SIRT‐1, NF‐κB, interferon‐gamma and interleukin 10. In conclusion, our results suggest that ETS induced the cardiac remodelling process. In addition, SM supplementation attenuated this process, along with oxidative stress reduction and energy metabolism modulation.


| INTRODUCTION
Exposure to tobacco smoke is considered the most important cause of preventable death in the world. 1 It is responsible for more than 6 million deaths per year and, according to some estimates, in 2020, it will cause more than 10 million deaths per year. 2 In the cardiovascular system, ETS is a known risk factor for atherosclerosis, endothelial dysfunction, acute coronary syndromes and sudden death. 3,4 However, some clinical and experimental studies evaluated the direct effects of tobacco smoke on the heart, independently of atherosclerosis or hypertension. [5][6][7][8] These studies showed that ETS induces cardiac hypertrophy, increases left cardiac chambers and leads to ventricular dysfunction. [9][10][11] These alterations are characteristics of the cardiac remodelling process. According to Cohn et al, 12 the cardiac remodelling may be defined as genome expression, molecular, cellular and interstitial changes that are manifested clinically as changes in size, shape and function of the heart after cardiac injury. In the acute phase of cardiac injury, remodelling is an adaptive process enabling the heart to maintain function; however, chronically it leads to heart failure and death. 12 Experimental studies showed that lipotoxicity, oxidative stress, inflammation, hemodynamic overload and increased expression of mitogen-activated protein kinases (MAPK) are important mechanistic pathways linked with the effects of tobacco smoke on cardiac remodelling. [13][14][15][16] To attenuate this process, the supplementation of antioxidant and anti-inflammatory substances, such as vitamin D, beta-carotene, retinoic acid and taurine, has been studied. [17][18][19][20] In this scenario, SM deserves attention due to its antioxidant and antiinflammatory properties.
Spondias mombin is a tropical fruit that belongs to the family Anacardiaceae. The fruit is a drupe of 3-4 cm in length, ovoid, oblong and flat at the base, having thin and smooth skin, a thin pulp, a yellow-orange colour and an acidic flavour. The fruit has a large white fibrous seed. 21 All parts of the plant (ie, leaves, pulp and skin) are reported to be useful and are rich in beta-cryptoxanthin, lutein, flavonoids and phenolic compounds. 22 This unique composition showed antioxidant and anti-inflammatory effects in clinical and experimental studies. [22][23][24] However, the effects of SM supplementation in cardiovascular health are not well studied. Akinmoladun et al 25 showed that supplementation with SM before cardiac injury induced by isoproterenol in rats protected against inflammation and oxidative stress. Interestingly, these effects were comparable with the effects of the angiotensin-converting enzyme inhibitor ramipril. 25 Despite these effects, there was no study investigating the effects of SM on cardiac remodelling induced by ETS. Thus, the objective of this study was to investigate the influence of SM supplementation on the cardiac remodelling process induced by ETS in rats.

| MATERIALS AND METHODS
This research protocol was approved by the Animal Ethics Committee of Botucatu Medical School (1116-2015), and it was performed in accordance with the National Institute of Health's Guide for the Care and Use of Laboratory Animals.
Male Wistar rats weighing 200-250 g were divided into 4 groups: group C (control, n = 20) comprised animals not exposed to cigarette smoke and received standard chow; group ETS (exposed to tobacco smoke, n = 20) comprised animals exposed to cigarette smoke and received standard chow; group ETS100 (n = 20) animals were exposed to cigarette smoke and received standard chow supplemented with 100 mg/kg body weight/d of SM; and group ETS250 (n = 20) animals were exposed to cigarette smoke and received standard chow supplemented with 250 mg/ kg body weight/d of SM. 25 The rats were housed in individual cages, in a temperature-controlled room (24°C) with a 12-hour light/dark cycle. Water was supplied ad libitum. The dietary intake was recorded daily. The animals were observed for 2 months, during which morphological, biochemical and functional analyses were performed.

| Chow preparation
The skin and pulp of SM were triturated, and the juice was maintained in a À80°C freezer. The total phenolic compounds, antioxidant activity and total carotenoids of the juice were analysed according to Kim

| Exposure to tobacco smoke
The ETS rats were exposed to cigarette smoke in a chamber (dimen-

| Isolated heart study: Langendorff preparation
After 2 months of cigarette exposure, 8 animals from each group were evaluated according to the previously described method. We registered the diastolic and systolic pressures, the maximum LV pressure decrease rate (ÀdP/dt) and the maximum LV pressure development rate (+dP/dt). Systolic function was evaluated by +dP/dt and diastolic function by ÀdP/dt. Developed pressure was also measured. The hearts that were subjected to the isolated heart study were not used for any other analysis, as retrograde perfusion can interfere with subsequent biochemical analysis. 33

| Morphometric analysis
The right and left ventricles (including the interventricular septum) were dissected and separated. Transverse sections of the LV were fixed in 10% buffered formalin and embedded in paraffin. Fivemicrometre-thick sections were stained with haematoxylin and eosin. The myocyte CSA was determined as previously described. 13

| Cardiac LH and antioxidant enzyme analysis
Left ventricle samples (100 mg) were homogenized in 5 mL of 0.1 mol/L cold sodium phosphate buffer, pH 7.4, containing 1 mmol/L ethylene diamine tetra-acetic acid. Tissue homogenates were prepared, and total protein concentration, glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) were assessed as previously specified. 27

| RESULTS
Only 1 animal (in the ETS250 group) died during the follow-up period. There was no difference among body weights in the beginning of the study; however, the groups exposed to cigarette smoke had lower body weight at the end of the experiment compared with the control group (Table 1). In addition, chow ingestion was lower in ani- of the isolated heart study were also similar among groups ( Table 2).
The morphological data are summarized in Table 3. LV/BW was higher in the ETS group compared with the C group. The animals in the ETS100 and ETS250 groups had values of LV/BW similar to C group. In addition, the CSA was lower in the C group compared with the ETS groups; however, ETS250 had lower values of CSA compared with the ETS group.
Oxidative stress data and antioxidant enzyme activity are shown in Table 4. The ETS group had higher cardiac levels of LH compared with the C group; and, ETS100 and ETS250 had lower concentrations of LH compared with the ETS group. The cardiac activity of CAT was lower in animals exposed to cigarette smoke. In addition, the activity of SOD and GSH-Px was lower in the ETS group compared with the C group, and the supplementation of SM increased these activities in the ETS100 and ETS250 groups compared with the ETS group (Table 4).
Regarding energy metabolism analysis, the data are presented in In the Western blot analysis, there were no differences in the expression of Nrf-2, SIRT-1, NF-jB, IFN-c, IL-10 and types I and III collagen among groups (Figures 1 and 2). CAT, catalase; ETS, exposed to tobacco smoke; GSH-Px, glutathione peroxidase; LH, lipid hydroperoxide; SOD, superoxide dismutase. Data are expressed as mean AE SD. One-way ANOVA/Tukey.

| DISCUSSION
The objective of our study was to analyse the influence of SM supplementation on the cardiac remodelling process induced by ETS in rats.
Our data showed that ETS induced the cardiac remodelling process.
In addition, SM supplementation attenuated this process, along with oxidative stress reduction and energy metabolism modulation.
Tobacco smoke can lead to cardiovascular diseases due to its effects on the vascular system and direct effects on cardiac remodelling. [5][6][7][8]38,39 In our study, as expected, ETS increased LV chamber | 4001 diameter and induced myocyte hypertrophy. However, we did not observe functional alterations in echocardiogram or in isolated heart analysis, as we previously reported. 10,11,14,15,17,18 Because the time of tobacco exposure was the same (2 months), we believe that alterations in cigarette content, in the last years, such as reduction in nicotine and tar content, could explain our results. Despite that, serum cotinine levels were increased in animals exposed to tobacco smoke. In addition, morphological and biochemical alterations were observed with our protocol. These alterations are characteristics of the cardiac remodelling process.
Due to the high socio-economic impact and the high mortality rates, it is relevant to identify factors that modulate the cardiac remodelling process. In this scenario, we can highlight the supplementation of substances with anti-inflammatory and antioxidant properties, such as SM. In our study, the chemical analysis we performed showed higher amounts of carotenoids, antioxidants and phenolic content in Nrf-2 is an important transcription factor that coordinates the expression of antioxidant response element-containing genes, including SOD, GSH-Px, heme oxygenase-1 and many enzymes involved in glutathione production. 41 It is interesting to observe that some studies suggest that the Nrf-2 pathway could also be stimulated by SIRT-1. 41,42 SIRT-1 is a nicotinamide adenosine dinucleotide (NAD + )-dependent protein deacetylase that possesses remarkable antioxidant capacity by deacetylating numerous substrates. Thus, these pathways are usually up-regulated to protect the organisms from ROS damage. 42 However, in our study, the expression of these proteins was not increased with ETS.
NFj-B is a key regulator in the inflammatory pathway, and its activation triggers the expression of downstream target genes, including inflammatory cytokines, chemokines and adhesion molecules. In our study, we also did not find alterations in pro-inflammatory cytokines or in the NFj-B expression, suggesting that inflammation is not as important as oxidative stress in this model of tobacco smoke exposure.
Alterations in energy metabolism were also important mechanisms in the cardiac remodelling process induced by tobacco exposure.
Under physiological conditions, free fatty acids are the main energy substrate of the heart, accounting for 60%-90% of the energy supply. 43,44 Fatty acids and glucose metabolites enter the citric acid cycle by b-oxidation and glycolysis, respectively, to generate FADH2 and NADH, which participate in the electron transport chain. In the pathological condition, the heart switches the fuel source from fatty acids to glucose, lactate and pyruvate. This shift can be observed through the increased activity of PFK, LDH and PD, enzymes of the glycolytic pathway, and the decreased activity of OHADH involved in the b-oxidative process. 43,44 We found these alterations in ETS rats. In addition, we also showed a reduction in energy production due to the decreased activity of complex I, II and ATP synthase. The SM supplementation decreased glycolysis and increased the b-oxidation and the oxidative phosphorylation. It is also important to observe that these effects were greater with the higher dose of SM supplementation.
In this study, we showed that SM supplementation, mainly at higher doses, attenuated the cardiac remodelling process induced by ETS. This effect was associated with reduction in oxidative damage and modulation of energy metabolism. We speculate that the synergic interaction between carotenoids, flavonoids and phenolic compounds could be responsible for these beneficial effects.
In conclusion, our results suggest that ETS induced the cardiac remodelling process. In addition, SM supplementation attenuated this process, along with oxidative stress reduction and energy metabo-