http://informahealthcare.com/ijf ISSN: 0963-7486 (print), 1465-3478 (electronic) Int J Food Sci Nutr, Early Online: 1–8 ! 2014 Informa UK Ltd. DOI: 10.3109/09637486.2014.893286
RESEARCH ARTICLE
Endogenous antioxidant defense induction by melon superoxide dismutase reduces cardiac hypertrophy in spontaneously hypertensive rats Julie Carillon1,2, Caroline Rugale3, Jean-Max Rouanet1, Jean-Paul Cristol1, Dominique Lacan2, and Bernard Jover3 Int J Food Sci Nutr Downloaded from informahealthcare.com by University of Laval on 06/24/14 For personal use only.
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UMR 204 – Pre´vention des Malnutritions et des Pathologies Associe´es, Universite´ Montpellier 2, Montpellier, France, 2Bionov S.A.R.L., Site Agroparc, Chemin des Meinajaries, Avignon, France, and 3Faculte´ de Pharmacie, EA 7288 (CPID), Montpellier, France
Abstract
Keywords
We assessed the influence of SODB, a melon superoxide dismutase (SOD), on left ventricular (LV) hypertrophy in SHR. SODB (4 or 40U SOD) was given orally for 4 or 28 days to SHR. For each treatment period, LV weight index (LVWI) and cardiomyocytes size were measured. SOD, glutathione peroxidase (GPx) and catalase expressions, and LV production and presence of superoxide anion were determined. Pro-inflammatory markers were also measured. SODB reduced LVWI and cardiomyocytes size after 4 or 28 days. Cardiac SOD and GPx increased by 30–40% with SODB. The presence but not production of superoxide anion was significantly reduced by SODB. No effect of SODB was detected on inflammatory status in any group. The beneficial effect of SODB on cardiac hypertrophy seems to be related to the stimulation of endogenous antioxidant defense, suggesting that SODB may be of interest as a dietary supplementation during conventional antihypertensive therapy.
Antioxidant enzymes, blood pressure, inflammation marker, superoxide anion
Introduction Left ventricular hypertrophy (LVH) is an adaptive response to pressure or volume overload that preserves cardiac function. LVH may also be considered as a strong predictor of cardiovascular events in clinical hypertension (Devereux & Alderman, 1993). In spontaneously hypertensive rats (SHR), a model of human essential hypertension, oxidative stress appears to be involved in the development and maintenance of hypertension and its associated cardiac alterations (Alvarez et al., 2008; Griendling et al., 2000). Reduction of reactive oxygen species (ROS) generation may thus have an important role in cardiac protection. For instance, apocynin, a specific NADPH oxidase inhibitor, reduced oxidative stress and suppressed cardiac hypertrophy beyond its anti-hypertensive effect in stroke-prone SHR (Yamamoto et al., 2006). Interestingly, treatment with an angiotensin-converting enzyme (ACE) inhibitor lowered mean arterial pressure, angiotensin II (AngII), and oxidative stress in SHR, suggesting that AngII is an essential factor in the maintenance of oxidative stress (Bolterman et al., 2005). AngII was reported to induce an overexpression of cytosolic proteins involved in the activation of NAD(P)H oxidase (Griendling et al., 1994; Rugale et al., 2005). Involvement of ROS in the effect of AngII was suggested by the beneficial influence of liposome-encapsulated superoxide dismutase (SOD) on vascular disease (Laursen et al., 1997) as well as the prevention of cardiac hypertrophy with the HMG-CoA reductase, simvastin (Delbosc et al., 2002). Correspondence: Bernard Jover, PhD, Faculte´ de Pharmacie, EA7288 (CPID), 15 Av Charles FLAHAULT, 34 093 Montpellier Cedex 5, France. Tel: (33) 411 75 95 02. Fax: (33) 475 11 95 47. E-mail: [email protected]
History Received 9 December 2013 Revised 17 January 2014 Accepted 26 January 2014 Published online 6 March 2014
Several antioxidants have beneficial effects on hypertension and associated organs damage (Mate et al., 2010; Zhou et al., 2012). Besides treatment with dietary antioxidants, an original way to increase antioxidant capacity could be by supplying antioxidant enzymes, which have longer lasting effects because of their lower rate of exhaustion than mere metabolites. SOD can be isolated from natural food and coated to protect its activity from digestive enzymes, thus allowing long-term oral treatment. We previously reported that SODB, a gastro-resistant encapsulatedmelon concentrate particularly rich in SOD, has antioxidant properties on animal models of obesity and atherosclerosis (De´corde´ et al., 2009, 2010), but its mechanisms of action are still unknown. We demonstrated, with in vitro tests, that the antioxidant capacity of this melon concentrate is due to its high content of SOD compared with the other extract compounds (Carillon et al., 2012). Moreover, an inhibitory effect on the ACE was observed in vitro with SODB (Carillon et al., 2012). These observations suggest that SODB could have beneficial effects against cardiovascular alterations. In this context, we assessed the influence of chronic administration of SODB on cardiac hypertrophy and blood pressure in a genetic model of hypertension, the SHR. Particularly, oxidative status, as well as inflammatory markers, were determined in cardiac tissue. Finally, the effect of SODB on Angiotensin I (AngI) conversion was evaluated and compared with the influence of the ACE inhibitor, enalapril.
Methods The present animal experiments complied with the European and French laws (permit numbers B-3417226 and 34179) and conform
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to the Guide for the Care and Use of Laboratory Animals published by the NIH (Publication No. 85-23, revised 1996). Preparation and characterization of SODB SOD by BionovÕ (SODB, Avignon, France) is a melon (not GMO) concentrate, particularly rich in SOD, resulting from a patented extraction process. For nutraceutical applications, SODB is coated in order to protect the SOD from digestive enzymes. In this study, it contains 14U SOD/mg powder measured according to Zhou & Prognon (2006).
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Animals and experimental design Seventy two 9-week-old SHR (Janvier, le Genest-St-Isle, France) were used in the present experiments. They were housed at 22 ± 1 C, subjected to a 12-h light/dark cycle with free access to both food (A04, SAFE, Augy, France) and tap water. Rats were assigned to four treatment groups (n ¼ 18 in each). Two groups received SODB at the daily dose of 4 or 40U SOD per os (mixed with food). The doses of SODB were chosen according to preliminary studies where 4U was the smaller dose being efficient, whereas 40U was the smaller dose with the maximal effect on cardiac mass and blood pressure (data not shown). A group received enalapril (Merck, Whitehouse Station, NJ) at a dose of 30 mg kg1 per day diluted in the drinking fluid (30 mg.100 mL1), whereas a group of untreated SHR served as controls. Treatments were given for 4 days or 28 days (n ¼ 6 or n ¼ 12 in each group). Rats were housed in individual cages and body weight, food and water consumptions and urine volume were measured daily throughout. Water balance (mL 24 h1) was calculated as the difference between daily water intake and urine volume and averaged for each rat over the 28-day period of observation. Tail-cuff pressure (TCP, Narco biosystems, Houston, TX) was recorded before and 2 days after the onset of treatments, and on day 7, 14, 21 and 28 in the 48 rats treated for 4 weeks. For each rat, TCP was expressed in absolute value (mmHg) as well as relative change from its basal, pre-treatment value (D%). At the end of the experimental period, rats were anesthetized (ketamine and xylazine, 75 and 25 mg/kg), and a polyethylene catheter (PE50) was inserted into the right carotid artery for arterial pressure and heart rate recording. In addition, the arterial pressure response to AngI bolus (50 ng) given i.v. (right jugular vein) was evaluated in rats receiving the treatments for 4 days only (n ¼ 6 in each of the four groups). Then, 5 mL of blood was sampled in all rats, and plasma and erythrocytes were separated by centrifugation (4000 rpm, 10 min) and stored at 80 C until analysis. Cardiac morphology Cardiac morphology was determined at 4 and 28 days of treatment. The left ventricle (LV) was weighed, and LV weight to body weight ratio was calculated (left ventricular weight index, LVWI). The LV was cut into several pieces for various determinations. One piece of LV was paraffin-embedded, cut (3 mm slices) and stained with hematoxylin-eosin. To determine the cardiomyocyte size, the shortest transverse diameter was measured in a blind fashion by three different observers on at least 40 transverse sections per heart using image analysis software (ImageJ, Bethesda, MD). All other parameters described below were determined at the end of the 28-day treatment period in the four groups (n ¼ 12 in each). Cardiac antioxidant enzymes expression by western blot The LV protein extraction was carried out on ice in 20 mM of Tris buffer (pH 6,8) containing 150 mM sodium chloride, 1 mM
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ethylenediaminetetraacetic acid (EDTA), 1% Triton 20%, 0.1% sodium dodecylsulfate (SDS), 1% protease inhibitor cocktail (P8340, Sigma-Aldrich, Saint Louis, MO). After centrifugation (1500 rpm, 15 min at 4 C), the supernatant was collected and extracted tissue proteins were then separated by sodium dodecylsulfate polyacrylamide gel electrophoresis. Equal amounts of proteins were loaded onto a 15% acrylamide gel with a 4% stacking acrylamide gel. Migration was conducted in a Trisglycine-SDS buffer (Sigma-Aldrich, Saint Louis, MO). After separation, proteins were transferred onto nitrocellulose membranes (Whatman, Dassel, Germany). Antioxidant proteins were detected by western blotting. The primary antibodies against rat Cu/Zn-SOD, Mn-SOD, GPx, CAT and the control protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased at R&D Systems (Minneapolis, MN). Expression of GAPDH was used for checking the equal protein load across gel tracks. The same secondary antibody from Invitrogen (Carlsbad, CA) coupled with alkaline phosphatase was used for revealing all the primary antibodies. Band densities were obtained by scanning the membranes. Image analysis (ImageJ) was used for quantification after standardization within membranes by expressing the density of each band of interest relative to that of GAPDH in the same lane. Results are then expressed as percent of values obtained in untreated SHR. Cardiac superoxide anion detection and production Dihydroethidine microphotography (DHE, Invitrogen, Eugene, OR) was used to detect superoxide anion in the LV. DHE penetrates the cells and it is oxidized by superoxide anion to form fluorescent products, which are, in turn, trapped by intercalation in the DNA. Sections were incubated with 2.5 mM of DHE at 37 C for 30 min in a humidified chamber protected from light. Fluorescence was visualized using TM300 microscope equipped with a digital imaging system (model DXM1200; Nikon, Champigny sur Marne, France), and quantified using image analysis software (ImageJ). LV production of superoxide anion was measured by lucigenin chemiluminescence as previously described (Sutra et al., 2007). Usually, the availability of superoxide anion for lucigenin depends on both production and scavenging. Here, cardiac Cu/Zn SOD was inhibited with the addition of 2 mM KCN to the supernatant (cytosol), and thus, only superoxide anion production was measured. The intensity of luminescence (BertholdTech CENTRO, Thoiry, France) was expressed as relative light units (RLU) per milligram of protein, determined with a commercial protein assay (Sigma, Saint Quentin Fallavier, France) according to the method given by Smith et al. (1985) and using bovine serum albumin as standard. Cardiac and plasma inflammatory factors Two hundred mg of LV tissue were homogenized in 2 mL of Tris buffer (10 mM, pH 7.4) containing 2 M NaCl, 1 mM EDTA, 0.01% Tween 80, 1 mM phenylmethylsulfonyl fluoride. After centrifugation (8400 rpm for 30 min at 4 C), the supernatant was used for cytokine determination. Levels of interleukin-1beta (IL-1b), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a) were assessed by ELISA using commercial kits from R&D Systems (Minneapolis, MN). Results were expressed as nanograms of interleukins per milligram of protein. Plasma concentration of IL-1b, IL-6 and TNF-a was also determined in the four experimental groups and expressed as picograms per milliliter. A piece of LV tissue was homogenized in a hypotonic buffer (pH 7.9) containing 20 mM HEPES, 10 mM EDTA, 10 mM KCl, 1% protease inhibitor cocktail, 0.1% dithiothreitol and 0.1%
Melon SOD reduces cardiac hypertrophy
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igepal. Nuclear extracts were then obtained by homogenizing pellet in a lysis buffer (pH 7.9) containing 20 mM HEPES, 1 mM EDTA, 200 mM sodium chloride, 10% glycerol, 1 mM dithiothreitol and 1% protease inhibitor cocktail. Nuclear factor-kappa B (NF-kB) activity was determined on nuclear extracts by ELISA using a commercial kit from Active motif (Rixensart, Belgium). Results were expressed as nanogram of NF-kB per milligram of protein. Statistical analyses
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Data (mean ± SEM) were analyzed by one-way analysis of variance or two-way analysis of variance for repeated measures when required (SOFA Statistics). Between groups differences were determined using the Fisher’s Protected Least Significant Difference test for multiple comparisons. Within-group differences were determined with the Student’s t-test for paired values. The level of significance was set for p50.05.
Results Blood pressure and cardiac morphology TCP was similar in all groups before the onset of treatments with a mean value of 192 ± 1 mmHg, n ¼ 48. During the 28 days of observation, TCP increased slowly in vehicle-treated SHR whereas it decreased rapidly and to the same extent with 4U and 40U of SODB. As expected, enalapril treatment was associated with a gradual and marked fall in blood pressure Table 1. Hemodynamic and cardiac characteristics of SHR after the 28 days of SODB administration.
Untreated BW (g) Water balance (mL/24 h) Final TCP (mmHg) Final SAP (mmHg) Response to 50 ng AngI (D mmHg) LVWI (mg HW/gBW)
Enalapril
SODB 4U
SODB 40 U
299 ± 7 24 ± 1
306 ± 3 27 ± 3
301 ± 5 27 ± 1
282 ± 4 26 ± 2
198 ± 5 203 ± 5 35 ± 5
143 ± 3* 145 ± 4* 15 ± 0*
180 ± 5* 189 ± 4* 40 ± 3
179 ± 4* 186 ± 2* 33 ± 6
2.61 ± 0.04 2.04 ± 0.04* 2.41 ± 0.04* 2.51 ± 0.04
Values are means ± SE. BW: body weight; TCP: tail-cuff pressure; SAP: systolic arterial pressure (carotid artery); AngI: angiotensin I. LVWI: left ventricular weight index; HW: heart weight. Water balance is expressed as mean value calculated over 28 days as the difference between water intake and urine volume. *p50.05 compared to untreated SHR.
Figure 1. Influence of long-term administration of SODB or enalapril on tail-cuff pressure in SHR. Results are expressed as relative changes from pre-treatment (Basal) blood pressure. *p50.05 compared to untreated SHR for each day of measure.
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(Table 1). As depicted in Figure 1 with the relative change in TCP, SODB induced a fall of 9 and 10% when compared to the final level achieved in untreated SHR. Enalapril was associated with a 25% decrease in TCP. Similarly, carotid arterial pressure measured in anesthetized rats was lowered by 7, 8 and 28%, respectively, in SODB 4U-, SODB 40U- and enalapril-treated SHR when compared to their untreated counterparts. The peak response to an exogenous bolus of AngI was reduced by approximately 60% in enalapril-treated SHR and no inhibition was detected in SHR receiving SODB (Table 1). LVWI determined after 4 days of treatment was significantly lower in rats receiving SODB at 4 and 40U or enalapril when compared to untreated SHR (2.45 ± 0.08, 2.54 ± 0.06 and 2.36 ± 0.06 versus 2.89 ± 0.03 mg HW. g1 BW, respectively). After 28 days of observation, LV mass index was further lowered in enalapril-treated SHR, while no additional reduction was detected with the two doses of SODB (Table 1). As presented in Figure 2, cardiomyocyte diameter was significantly reduced by SODB from the fourth day. Water intake and excretion were stable in the untreated SHR while a parallel rise was observed for both parameters in the three treated groups (data not shown). Thus, water balance was comparable in all groups (Table 1). Oxidative status As shown in Figure 3 the two doses of SODB induced a significant increase in the cardiac quantity of total SOD and GPx after 28 days of treatment (by 30–40% compared to untreated SHR), while enalapril had no effect on these expressions. CAT expression was not modulated in any group. As shown in Figure 4, DHE intensity was significantly reduced by the two doses of SODB with an average reduction of 13% compared to untreated SHR. No significant difference was detected in cardiac superoxide anion production in SODB- and enalapril-treated SHR. Inflammatory status None of the treatments affected cardiac concentration of the nuclear factor NF-kB. Moreover, no difference was observed in left ventricular IL-6, IL-1b or TNF-a concentrations in any group (Table 2). Plasma concentration of IL-6, IL-1b or TNF-a was similar in untreated- and SODB-treated SHR. Enalapril administration was associated with a reduction in the three markers and significance was only achieved for TNF-a (Table 2).
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Figure 2. Influence of short- (4 days) and long- (28 days) term treatment with SODB or enalapril on cardiomyocyte size. Upper panel: Cardiomyocyte staining with hematoxylin-eosin at the end of observation period (bar ¼ 25 mm). Lower panel: Cardiomyocyte size determined on at least 40 transverse sections per heart in 6 and 12 rats per group for the short- and long-term treatment, respectively. *p50.05 compared to untreated SHR for 4 and 28 days of treatment.
Discussion The present study demonstrates that genetically hypertensive rats treated with SODB have a reduced cardiac mass. The lowering of cardiac mass was equated with the LVWI and confirmed very clearly by direct measurement of cardiomyocytes diameter on tissue slices. The effect of SODB was observed similarly with the two doses used in the study (4 or 40 U SOD/day). The cardiac effect was detected since the first measurement, i.e. after 4 days of treatment, and was maintained after the 4 weeks of SODB administration. Among the various parameters which could participate in the reduction of cardiac mass is the lowering of preload. During the 28 days of treatment, neither water intake nor urine volume was modified by SODB, while both parameters increased to the same extent during enalapril administration. Although no direct measurement of blood volume or venous return was performed in the present experiment, the lack of change in water handling does not favor a major involvement of preload reduction in the beneficial cardiac influence of the melon SOD.
A reduction of afterload may also partly explain the cardiac effect of SODB administration. Yet, the decrease in arterial pressure was slight as measured indirectly in conscious rats or directly recorded in anesthetized animals. As observed on cardiac mass, the decrease in arterial blood pressure was similar with the two doses of SODB. In addition, the peak effect of SODB on blood pressure was achieved after four days of treatment and remained stable throughout. The slight antihypertensive influence of SODB might be related to its ACE inhibitor activity previously reported in vitro using hippuryl-L-histidyl-L-leucine as the substrate (Carillon et al., 2012). In order to investigate such an effect, the pressure response to exogenous AngI administration was evaluated in rats treated for 4 days only, when the blood pressure and cardiac mass lowering effect of SODB were already maximal. In contrast to that observed with enalapril, the peak response was not affected by SODB treatment even at the highest dose. Although we cannot exclude a very early effect of SODB particularly in cardiac tissue, the inhibition of ACE does not seem to play a major role in the effect of SODB in vivo. Interestingly,
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DOI: 10.3109/09637486.2014.893286
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Figure 3. Influence of long-term administration of SODB or enalapril on cardiac antioxidant defense expression. (A) Cardiac total SOD expression (Cu/Zn-SOD and Mn-SOD). (B) Cardiac GPx expression. (C) Cardiac CAT expression. Quantification was made after standardization within membranes by expressing the density of each band of interest relative to that of GAPDH in the same lane. Results are then expressed as relative changes from untreated SHR band intensity. *p50.05 compared to untreated SHR.
blood pressure reduction was weaker with SODB than enalapril (5 versus 20%, approximately), while cardiac hypertrophy was reduced to the same extent with the two compounds. A reduction in arterial pressure is not necessarily required for the beneficial effect of treatments on cardiac hypertrophy. A non-antihypertensive dose of enalapril in SHR (Piotrkowski et al., 2009) or ramipril in aortic banding hypertension (Linz et al., 1992) prevent cardiac tissue remodeling. A non-angiotensin-related compound such as resveratrol also prevents the development of cardiac hypertrophy in the SHR independently of blood pressure reduction (Thandapilly et al., 2010). Conversely, hydralazine administration has no effect on cardiac mass despite a marked reduction of blood pressure in the SHR (Gupta et al., 2005). Altogether, these findings indicate that the beneficial effect on cardiac hypertrophy cannot be entirely explained by reduction in pressure or volume overload, and suggest that SODB has an additional influence independently of the circulating renin–angiotensin system. In the current study, none of the two doses of SODB reduced NF-kB or the pro-inflammatory cytokines concentrations in cardiac tissue. In addition, SODB did not influence circulating cytokines. Although a very early effect cannot be ruled out, our results do not favor a major involvement of an anti-inflammatory effect in the beneficial influence of SODB. Besides hemodynamic changes or anti-inflammatory influence, reduction in the oxidative status appears to be an important
mechanism of the antihypertrophic effect of SODB. No significant change in chemiluminescence intensity using lucigenin probe was detected. Although our present observation must be confirmed on unfixed frozen tissue, a slight reduction in the staining by DHE of formalin-fixed, paraffin-embedded cardiac tissue was observed in rats treated by SODB. If we assume that lucigenin (in the presence of KCN) provides a chemiluminescent substrate to monitor superoxide production by NADPH oxidase and that DHE is a reliable marker of O2 intracellular presence, it is suggested that O2 scavenging rather than formation was mainly involved in the antihypertrophic effect of SODB in our setting. Interestingly, SODB administration was associated with an increased expression of endogenous antioxidant defenses (SOD and GPx). Potent induction of cellular antioxidant enzymes by grape seed polyphenols (Du et al., 2007) was previously reported in cultured rat cardiac H9C2 cells. Zhou et al. (2012) have also shown that osthol, a natural coumarin compound, could induce endogenous SOD and GPx in hypertrophied heart of rats. In recent studies, we have shown that SODB supplementation for 28 days could increase endogenous antioxidant enzymes (SOD, GPx and CAT) in the liver and adipose tissue of obese hamsters (Carillon et al., 2013b,c), and in the liver of healthy rats (Carillon et al., 2013a). Even if SODB was coated in order to reach intestinal mucosa, SOD protein is probably not absorbed. The question arises of how an exogenous melon SOD administration, could induce endogenous antioxidant defense?
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Figure 4. Influence of 28 days of treatment with SODB or enalapril on dihydroethidine staining. Upper panel: Example of DHE staining of left ventricle slices at the end of observation period ( 100). Lower panel: DHE intensity size determined on at least 10 transverse sections per heart. *p50.05 compared to untreated SHR.
Table 2. Oxidative and inflammatory status of the heart of SHR after the 28 days of SODB administration.
Untreated
Enalapril
SODB 4U
SODB 40 U
Cardiac O2 detection by Lucigenin 9420 ± 1523 6302 ± 398 8249 ± 592 6209 ± 821 (RLU/mg prot) Cardiac concentration (ng/mg prot) IL-1b 5.9 ± 0.7 IL-6 11.8 ± 3.2 TNF-a 7.5 ± 1.3 NF-kB 50.6 ± 8.6
6.1 ± 0.9 14.3 ± 2.4 8.7 ± 1.3 38.5 ± 4.6
6.7 ± 1.2 6.8 ± 0.6 11.7 ± 4.0 12.0 ± 3.1 8.8 ± 1.5 9.8 ± 1.0 50.3 ± 11.2 36.7 ± 8.2
Plasma concentration (pg/mL) IL-1b 76 ± 8 IL-6 408 ± 79 TNF-a 112 ± 6
57 ± 6 335 ± 49 76 ± 9*
102 ± 34 464 ± 33 81 ± 14
109 ± 26 452 ± 50 91 ± 17
Values are means ± SE. RLU: relative light units; IL-1b: interleukin1beta; IL-6: interleukin-6; TNF-a: tumor necrosis factor-alpha; NF-kB: nuclear factor-kappaB. *p50.05 compared to untreated SHR.
Other exogenous SODs seem to act by inducing endogenous antioxidant defense, as reviewed by Carillon et al. (2013d), and some hypothesis have been proposed even if the precise mechanism is still unknown. Indeed, the nuclear factor-E2-related factor (Nrf2)/antioxidant response element (ARE) pathway (Li et al., 2012), may be involved in the induction of endogenous antioxidant enzymes by SOD. The higher amount of endogenous antioxidant enzymes in cardiac tissue and probably the subsequent reduction of cardiac oxidative stress could explain the anti-hypertrophic and antihypertensive effects of SODB. An influence of antioxidant defense on cardiac morphology was previously suggested by the reduction of both Mn-SOD and CuZn-SOD in the hypertrophied heart of aged SHR (Ito et al., 1995). In mice, SOD deficiency was associated with a more marked LVH after myocardial infarction (van Deel et al., 2008). SOD and GPx deficiency was also observed in the blood of patients with permanent essential hypertension (Iarema et al., 1992). On the other hand, treatment with tempol, a SOD mimetic, had antihypertensive effect on SHR (Peixoto et al., 2009) and attenuated cardiac hypertrophy in GLUT4-knockout insulin-resistant mice (Ritchie et al., 2007). Consistent with the latter study using tempol
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DOI: 10.3109/09637486.2014.893286
(Ritchie et al., 2007), SODB induced a reduction in cardiac hypertrophy even at a dose (4U) insufficient to lower cardiac superoxide anion production. Since a concomitant stimulation of SOD and GPx was observed, the low dose of SODB probably also acted through the blunting of oxidative stress by subsequent endogenous antioxidant enzymes. Heart growth associated with pathological situation implicates a large number of signaling pathways (see Heineke & Molkentin, 2006 for review), where oxidative stress and redox signaling had probably a substantial role (review by Seddon et al., 2007). Which are the precise molecular targets of the cardiovascular effects of SODB remain to be investigated in future dedicated experiments. The induction of endogenous antioxidant defense after SODB administration could explain why high dose (40U) and long-term supplementation (28 days) did not exert more benefits. Our results suggest that a supplementation of 4 days with 4U of SODB is sufficient to induce a stock of endogenous antioxidant enzymes. In addition, other doses (10 and 80U) have been assessed in the same conditions, and no more beneficial effects have been observed (data not shown). Moreover, the induction of several antioxidant enzymes avoids imbalance, which could be involved in some diseases such as Down syndrome (Muchova et al., 2001; Perluigi & Butterfield, 2012). Indeed, GPx removes H2O2 produced after dismutation of O2 by SOD.
Conclusions In summary, we have shown that SODB has a beneficial effect on cardiac hypertrophy in a model of genetic hypertension. Such an effect was related to a reduction of oxidative stress through stimulation of endogenous antioxidant defense in cardiac tissue rather than hemodynamic or anti-inflammatory influence. These findings suggest that SODB could play an important role in cardiac protection in essential hypertension. Its beneficial influence as a dietary supplementation during conventional antihypertensive therapy has to be investigated. In addition, the effect of SODB on other tissues and/or different pathologies that involve oxidative stress deserves further investigations.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. Julie Carillon was supported by a ‘‘CIFRE grant’’ (Convention Industrielle de Formation par la REcherche, n 0417/2010) from Bionov (Avignon, France) and the French ‘‘Association Nationale de la Recherche et de la Technologie’’. Dominique Lacan is Bionov R&D director.
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activity of a melon concentrate rich in superoxide dismutase. Food Chem 135:1298–1302. Carillon J, Romain C, Bardy G, Fouret G, Feillet-Coudray C, Gaillet S, Lacan D, et al. 2013b. Cafeteria diet induces obesity and insulin resistance associated with oxidative stress but not with inflammation. Improvement by dietary supplementation of a melon superoxide dismutase. Free Radic Biol Med 65:254–261. Carillon J, Rouanet JM, Cristol JP, Brion R. 2013d. Superoxide dismutase administration, a potential therapy against oxidative stress related diseases: several routes of supplementation and proposal of an original mechanism of action. Pharm Res 30:2718–2728. De´corde´ K, Agne A, Lacan D, Ramos J, Fouret G, Ventura E, Feilletcoudray C, et al. 2009. Preventive effect of a melon extract rich in superoxide scavenging activity on abdominal and liver fat and adipokine imbalance in high-fat-fed hamsters. J Agric Food Chem 57:6461–6467. De´corde´ K, Ventura E, Lacan D, Ramos J, Cristol JP, Rouanet JM. 2010. An SOD rich melon extract ExtramelÕ prevents aortic lipids and liver steatosis in diet-induced model of atherosclerosis. Nutr Metabol Cardiovasc Dis 20:301–307. Delbosc S, Cristol JP, Descomps B, Mimran A, Jover B. 2002. Simvastatin prevents angiotensin II-induced cardiac alterations and oxidative stress. Hypertension 40:142–147. Devereux RB, Alderman MH. 1993. Role of preclinical cardiovascular disease in the evolution from risk factor exposure to development of morbid events. Circulation 88:1444–1455. Du Y, Guo H, Lou H. 2007. Grape seed polyphenols protect cardiac cells from apoptosis via induction of endogenous antioxidant enzymes. J Agric Food Chem 55:1695–1701. Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. 1994. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res 74:1141–1148. Griendling KK, Sorescu D, Ushio-Fukai M. 2000. NAD(P)H oxidase role in cardiovascular biology and disease. Circ Res 86:494–501. Gupta S, Young D, Sen S. 2005. Inhibition of NF-kappaB induces regression of cardiac hypertrophy, independent of blood pressure control, in spontaneously hypertensive rats. Am J Physiol Heart Circul Physiol 289:H20–H29. Heineke J, Molkentin JD. 2006. Regulation of cardiac hypertrophy by intracellular signalling pathways. Nature Rev Mol Cell Biol 7:589–600. Iarema N, Konovalova GG, Lankin VZ. 1992. Changes in the activity of antioxidant enzymes in patients with hypertension. Kardiologia 32: 46–48. Ito H, Torii M, Suzuki T. 1995. Decrease superoxide-dismutase activity and increased superoxide anion production in cardiac-hypertrophy of spontaneously hypertensive rats. Clin Exp Hypertens 17:803–816. Laursen JB, Rajagopalan S, Galis Z, Tarpey M, Freeman BA, Harrison DG. 1997. Role of superoxide in angiotensin II-induced but not catecholamine-induced hypertension. Circulation 95:588–593. Li H, Xia N, Fo¨rstermann U. 2012. Cardiovascular effects and molecular targets of resveratrol. Nitric Oxide. 26:102–110. Linz W, Schaper J, Wiemer G, Albus U, Scho¨lkens BA. 1992. Ramipril prevents left ventricular hypertrophy with myocardial fibrosis without blood pressure reduction: a one year study in rats. Br J Pharmacol 107: 970–975. Mate A, Miguel-Carrasco JL, Monserrat MT, Vazquez CM. 2010. Systemic antioxidant properties of L-carnitine in two different models of arterial hypertension. J Physiol Biochem 66:127–136. Muchova J, Sustrova M, Garaiova I, Liptakova A, Blazicek P, Kvasnicka P, Pueschel S, Durackova Z. 2001. Influence of age on activities of antioxidant enzymes and lipid peroxidation products in erythrocytes and neutrophils of Down syndrome patients. Free Radic Biol Med 31: 499–508. Peixoto EBMI, Pessoa BS, Biswas SK, de Faria JBL. 2009. Antioxidant SOD mimetic prevents NADPH oxidase-induced oxidative stress and renal damage in the early stage of experimental diabetes and hypertension. Am J Nephrol 29:309–318. Perluigi M, Butterfield A. 2012. Oxidative stress and Down syndrome: a route toward Alzheimer-like dementia. Curr Gerontol Geriatr Res 2012:724904. doi: 10.1155/2012/724904. Piotrkowski B, Koch OR, De Cavanagh EM, Fraga CG. 2009. Cardiac mitochondrial function and tissue remodelling are improved by a nonantihypertensive dose of enalapril in spontaneously hypertensive rats. Free Radic Res 43:390–399. Ritchie RH, Quinn JM, Cao AH, Drummond GR, Kaye DM, Favaloro JM, Proiettoe J, Delbridgeb LMD. 2007. The antioxidant tempol inhibits
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cardiac hypertrophy in the insulin-resistant GLUT4-deficient mouse in vivo. J Mol Cell Cardiol 42:1119–1128. Rugale C, Cordaillat M, Mimran A, Jover B. 2005. Prevention and reversal by enalapril of target organ damage in angiotensin II hypertension. J Renin-Angio-Aldo S 6:154–160. Seddon M, Looi YH, Shah AM. 2007. Oxidative stress and redox signalling in cardiac hypertrophy and heart failure. Heart 93:903–907. Smith SK, Krohn RI, Mallia AK, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DK. 1985. Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85. Sutra T, De´corde´ K, Riss J, Dallas C, Cristol JP, Rouanet JM. 2007. A commercial extract of fruits and vegetables, Oxynea, acts as a powerful antiatherosclerotic supplement in an animal model by reducing cholesterolemia, oxidative stress, and NADPH oxidase expression. J Agric Food Chem 55:4258–4263. Thandapilly SJ, Wojciechowski P, Behbahani J, Louis XL, Yu L, Juric D, Kopilas MA, et al. 2010. Resveratrol prevents the development of
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pathological cardiac hypertrophy and contractile dysfunction in the SHR without lowering blood pressure. Am J Hypertens 23:192–196. van Deel ED, Lu Z, Xu X, Zhu G, Hu X, Oury TD, Bache RJ, et al. 2008. Extracellular superoxide dismutase protects the heart against oxidative stress and hypertrophy after myocardial infarction. Free Rad Biol Med 44:1305–1313. Yamamoto E, Lai ZF, Yamashita T, Tanaka T, Kataoka K, Tokutomi Y, Ito T, et al. 2006. Enhancement of cardiac oxidative stress by tachycardia and its critical role in cardiac hypertrophy and fibrosis. J Hypertens 24:2054–2069. Zhou F, Zhong W, Xue J, Gu ZL, Xie ML. 2012. Reduction of rat cardiac hypertrophy by osthol is related to regulation of cardiac oxidative stress and lipid metabolism. Lipids 47:987–994. Zhou JY, Prognon P. 2006. Raw material enzymatic activity determination: a specific case for validation and comparison of analytical methods – the example of superoxide dismutase (SOD). J Pharm Biomed Analysis 40:1143–1148.
RESEARCH ARTICLE
Endogenous antioxidant defense induction by melon superoxide dismutase reduces cardiac hypertrophy in spontaneously hypertensive rats Julie Carillon1,2, Caroline Rugale3, Jean-Max Rouanet1, Jean-Paul Cristol1, Dominique Lacan2, and Bernard Jover3 Int J Food Sci Nutr Downloaded from informahealthcare.com by University of Laval on 06/24/14 For personal use only.
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UMR 204 – Pre´vention des Malnutritions et des Pathologies Associe´es, Universite´ Montpellier 2, Montpellier, France, 2Bionov S.A.R.L., Site Agroparc, Chemin des Meinajaries, Avignon, France, and 3Faculte´ de Pharmacie, EA 7288 (CPID), Montpellier, France
Abstract
Keywords
We assessed the influence of SODB, a melon superoxide dismutase (SOD), on left ventricular (LV) hypertrophy in SHR. SODB (4 or 40U SOD) was given orally for 4 or 28 days to SHR. For each treatment period, LV weight index (LVWI) and cardiomyocytes size were measured. SOD, glutathione peroxidase (GPx) and catalase expressions, and LV production and presence of superoxide anion were determined. Pro-inflammatory markers were also measured. SODB reduced LVWI and cardiomyocytes size after 4 or 28 days. Cardiac SOD and GPx increased by 30–40% with SODB. The presence but not production of superoxide anion was significantly reduced by SODB. No effect of SODB was detected on inflammatory status in any group. The beneficial effect of SODB on cardiac hypertrophy seems to be related to the stimulation of endogenous antioxidant defense, suggesting that SODB may be of interest as a dietary supplementation during conventional antihypertensive therapy.
Antioxidant enzymes, blood pressure, inflammation marker, superoxide anion
Introduction Left ventricular hypertrophy (LVH) is an adaptive response to pressure or volume overload that preserves cardiac function. LVH may also be considered as a strong predictor of cardiovascular events in clinical hypertension (Devereux & Alderman, 1993). In spontaneously hypertensive rats (SHR), a model of human essential hypertension, oxidative stress appears to be involved in the development and maintenance of hypertension and its associated cardiac alterations (Alvarez et al., 2008; Griendling et al., 2000). Reduction of reactive oxygen species (ROS) generation may thus have an important role in cardiac protection. For instance, apocynin, a specific NADPH oxidase inhibitor, reduced oxidative stress and suppressed cardiac hypertrophy beyond its anti-hypertensive effect in stroke-prone SHR (Yamamoto et al., 2006). Interestingly, treatment with an angiotensin-converting enzyme (ACE) inhibitor lowered mean arterial pressure, angiotensin II (AngII), and oxidative stress in SHR, suggesting that AngII is an essential factor in the maintenance of oxidative stress (Bolterman et al., 2005). AngII was reported to induce an overexpression of cytosolic proteins involved in the activation of NAD(P)H oxidase (Griendling et al., 1994; Rugale et al., 2005). Involvement of ROS in the effect of AngII was suggested by the beneficial influence of liposome-encapsulated superoxide dismutase (SOD) on vascular disease (Laursen et al., 1997) as well as the prevention of cardiac hypertrophy with the HMG-CoA reductase, simvastin (Delbosc et al., 2002). Correspondence: Bernard Jover, PhD, Faculte´ de Pharmacie, EA7288 (CPID), 15 Av Charles FLAHAULT, 34 093 Montpellier Cedex 5, France. Tel: (33) 411 75 95 02. Fax: (33) 475 11 95 47. E-mail: [email protected]
History Received 9 December 2013 Revised 17 January 2014 Accepted 26 January 2014 Published online 6 March 2014
Several antioxidants have beneficial effects on hypertension and associated organs damage (Mate et al., 2010; Zhou et al., 2012). Besides treatment with dietary antioxidants, an original way to increase antioxidant capacity could be by supplying antioxidant enzymes, which have longer lasting effects because of their lower rate of exhaustion than mere metabolites. SOD can be isolated from natural food and coated to protect its activity from digestive enzymes, thus allowing long-term oral treatment. We previously reported that SODB, a gastro-resistant encapsulatedmelon concentrate particularly rich in SOD, has antioxidant properties on animal models of obesity and atherosclerosis (De´corde´ et al., 2009, 2010), but its mechanisms of action are still unknown. We demonstrated, with in vitro tests, that the antioxidant capacity of this melon concentrate is due to its high content of SOD compared with the other extract compounds (Carillon et al., 2012). Moreover, an inhibitory effect on the ACE was observed in vitro with SODB (Carillon et al., 2012). These observations suggest that SODB could have beneficial effects against cardiovascular alterations. In this context, we assessed the influence of chronic administration of SODB on cardiac hypertrophy and blood pressure in a genetic model of hypertension, the SHR. Particularly, oxidative status, as well as inflammatory markers, were determined in cardiac tissue. Finally, the effect of SODB on Angiotensin I (AngI) conversion was evaluated and compared with the influence of the ACE inhibitor, enalapril.
Methods The present animal experiments complied with the European and French laws (permit numbers B-3417226 and 34179) and conform
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to the Guide for the Care and Use of Laboratory Animals published by the NIH (Publication No. 85-23, revised 1996). Preparation and characterization of SODB SOD by BionovÕ (SODB, Avignon, France) is a melon (not GMO) concentrate, particularly rich in SOD, resulting from a patented extraction process. For nutraceutical applications, SODB is coated in order to protect the SOD from digestive enzymes. In this study, it contains 14U SOD/mg powder measured according to Zhou & Prognon (2006).
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Animals and experimental design Seventy two 9-week-old SHR (Janvier, le Genest-St-Isle, France) were used in the present experiments. They were housed at 22 ± 1 C, subjected to a 12-h light/dark cycle with free access to both food (A04, SAFE, Augy, France) and tap water. Rats were assigned to four treatment groups (n ¼ 18 in each). Two groups received SODB at the daily dose of 4 or 40U SOD per os (mixed with food). The doses of SODB were chosen according to preliminary studies where 4U was the smaller dose being efficient, whereas 40U was the smaller dose with the maximal effect on cardiac mass and blood pressure (data not shown). A group received enalapril (Merck, Whitehouse Station, NJ) at a dose of 30 mg kg1 per day diluted in the drinking fluid (30 mg.100 mL1), whereas a group of untreated SHR served as controls. Treatments were given for 4 days or 28 days (n ¼ 6 or n ¼ 12 in each group). Rats were housed in individual cages and body weight, food and water consumptions and urine volume were measured daily throughout. Water balance (mL 24 h1) was calculated as the difference between daily water intake and urine volume and averaged for each rat over the 28-day period of observation. Tail-cuff pressure (TCP, Narco biosystems, Houston, TX) was recorded before and 2 days after the onset of treatments, and on day 7, 14, 21 and 28 in the 48 rats treated for 4 weeks. For each rat, TCP was expressed in absolute value (mmHg) as well as relative change from its basal, pre-treatment value (D%). At the end of the experimental period, rats were anesthetized (ketamine and xylazine, 75 and 25 mg/kg), and a polyethylene catheter (PE50) was inserted into the right carotid artery for arterial pressure and heart rate recording. In addition, the arterial pressure response to AngI bolus (50 ng) given i.v. (right jugular vein) was evaluated in rats receiving the treatments for 4 days only (n ¼ 6 in each of the four groups). Then, 5 mL of blood was sampled in all rats, and plasma and erythrocytes were separated by centrifugation (4000 rpm, 10 min) and stored at 80 C until analysis. Cardiac morphology Cardiac morphology was determined at 4 and 28 days of treatment. The left ventricle (LV) was weighed, and LV weight to body weight ratio was calculated (left ventricular weight index, LVWI). The LV was cut into several pieces for various determinations. One piece of LV was paraffin-embedded, cut (3 mm slices) and stained with hematoxylin-eosin. To determine the cardiomyocyte size, the shortest transverse diameter was measured in a blind fashion by three different observers on at least 40 transverse sections per heart using image analysis software (ImageJ, Bethesda, MD). All other parameters described below were determined at the end of the 28-day treatment period in the four groups (n ¼ 12 in each). Cardiac antioxidant enzymes expression by western blot The LV protein extraction was carried out on ice in 20 mM of Tris buffer (pH 6,8) containing 150 mM sodium chloride, 1 mM
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ethylenediaminetetraacetic acid (EDTA), 1% Triton 20%, 0.1% sodium dodecylsulfate (SDS), 1% protease inhibitor cocktail (P8340, Sigma-Aldrich, Saint Louis, MO). After centrifugation (1500 rpm, 15 min at 4 C), the supernatant was collected and extracted tissue proteins were then separated by sodium dodecylsulfate polyacrylamide gel electrophoresis. Equal amounts of proteins were loaded onto a 15% acrylamide gel with a 4% stacking acrylamide gel. Migration was conducted in a Trisglycine-SDS buffer (Sigma-Aldrich, Saint Louis, MO). After separation, proteins were transferred onto nitrocellulose membranes (Whatman, Dassel, Germany). Antioxidant proteins were detected by western blotting. The primary antibodies against rat Cu/Zn-SOD, Mn-SOD, GPx, CAT and the control protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased at R&D Systems (Minneapolis, MN). Expression of GAPDH was used for checking the equal protein load across gel tracks. The same secondary antibody from Invitrogen (Carlsbad, CA) coupled with alkaline phosphatase was used for revealing all the primary antibodies. Band densities were obtained by scanning the membranes. Image analysis (ImageJ) was used for quantification after standardization within membranes by expressing the density of each band of interest relative to that of GAPDH in the same lane. Results are then expressed as percent of values obtained in untreated SHR. Cardiac superoxide anion detection and production Dihydroethidine microphotography (DHE, Invitrogen, Eugene, OR) was used to detect superoxide anion in the LV. DHE penetrates the cells and it is oxidized by superoxide anion to form fluorescent products, which are, in turn, trapped by intercalation in the DNA. Sections were incubated with 2.5 mM of DHE at 37 C for 30 min in a humidified chamber protected from light. Fluorescence was visualized using TM300 microscope equipped with a digital imaging system (model DXM1200; Nikon, Champigny sur Marne, France), and quantified using image analysis software (ImageJ). LV production of superoxide anion was measured by lucigenin chemiluminescence as previously described (Sutra et al., 2007). Usually, the availability of superoxide anion for lucigenin depends on both production and scavenging. Here, cardiac Cu/Zn SOD was inhibited with the addition of 2 mM KCN to the supernatant (cytosol), and thus, only superoxide anion production was measured. The intensity of luminescence (BertholdTech CENTRO, Thoiry, France) was expressed as relative light units (RLU) per milligram of protein, determined with a commercial protein assay (Sigma, Saint Quentin Fallavier, France) according to the method given by Smith et al. (1985) and using bovine serum albumin as standard. Cardiac and plasma inflammatory factors Two hundred mg of LV tissue were homogenized in 2 mL of Tris buffer (10 mM, pH 7.4) containing 2 M NaCl, 1 mM EDTA, 0.01% Tween 80, 1 mM phenylmethylsulfonyl fluoride. After centrifugation (8400 rpm for 30 min at 4 C), the supernatant was used for cytokine determination. Levels of interleukin-1beta (IL-1b), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a) were assessed by ELISA using commercial kits from R&D Systems (Minneapolis, MN). Results were expressed as nanograms of interleukins per milligram of protein. Plasma concentration of IL-1b, IL-6 and TNF-a was also determined in the four experimental groups and expressed as picograms per milliliter. A piece of LV tissue was homogenized in a hypotonic buffer (pH 7.9) containing 20 mM HEPES, 10 mM EDTA, 10 mM KCl, 1% protease inhibitor cocktail, 0.1% dithiothreitol and 0.1%
Melon SOD reduces cardiac hypertrophy
DOI: 10.3109/09637486.2014.893286
igepal. Nuclear extracts were then obtained by homogenizing pellet in a lysis buffer (pH 7.9) containing 20 mM HEPES, 1 mM EDTA, 200 mM sodium chloride, 10% glycerol, 1 mM dithiothreitol and 1% protease inhibitor cocktail. Nuclear factor-kappa B (NF-kB) activity was determined on nuclear extracts by ELISA using a commercial kit from Active motif (Rixensart, Belgium). Results were expressed as nanogram of NF-kB per milligram of protein. Statistical analyses
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Data (mean ± SEM) were analyzed by one-way analysis of variance or two-way analysis of variance for repeated measures when required (SOFA Statistics). Between groups differences were determined using the Fisher’s Protected Least Significant Difference test for multiple comparisons. Within-group differences were determined with the Student’s t-test for paired values. The level of significance was set for p50.05.
Results Blood pressure and cardiac morphology TCP was similar in all groups before the onset of treatments with a mean value of 192 ± 1 mmHg, n ¼ 48. During the 28 days of observation, TCP increased slowly in vehicle-treated SHR whereas it decreased rapidly and to the same extent with 4U and 40U of SODB. As expected, enalapril treatment was associated with a gradual and marked fall in blood pressure Table 1. Hemodynamic and cardiac characteristics of SHR after the 28 days of SODB administration.
Untreated BW (g) Water balance (mL/24 h) Final TCP (mmHg) Final SAP (mmHg) Response to 50 ng AngI (D mmHg) LVWI (mg HW/gBW)
Enalapril
SODB 4U
SODB 40 U
299 ± 7 24 ± 1
306 ± 3 27 ± 3
301 ± 5 27 ± 1
282 ± 4 26 ± 2
198 ± 5 203 ± 5 35 ± 5
143 ± 3* 145 ± 4* 15 ± 0*
180 ± 5* 189 ± 4* 40 ± 3
179 ± 4* 186 ± 2* 33 ± 6
2.61 ± 0.04 2.04 ± 0.04* 2.41 ± 0.04* 2.51 ± 0.04
Values are means ± SE. BW: body weight; TCP: tail-cuff pressure; SAP: systolic arterial pressure (carotid artery); AngI: angiotensin I. LVWI: left ventricular weight index; HW: heart weight. Water balance is expressed as mean value calculated over 28 days as the difference between water intake and urine volume. *p50.05 compared to untreated SHR.
Figure 1. Influence of long-term administration of SODB or enalapril on tail-cuff pressure in SHR. Results are expressed as relative changes from pre-treatment (Basal) blood pressure. *p50.05 compared to untreated SHR for each day of measure.
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(Table 1). As depicted in Figure 1 with the relative change in TCP, SODB induced a fall of 9 and 10% when compared to the final level achieved in untreated SHR. Enalapril was associated with a 25% decrease in TCP. Similarly, carotid arterial pressure measured in anesthetized rats was lowered by 7, 8 and 28%, respectively, in SODB 4U-, SODB 40U- and enalapril-treated SHR when compared to their untreated counterparts. The peak response to an exogenous bolus of AngI was reduced by approximately 60% in enalapril-treated SHR and no inhibition was detected in SHR receiving SODB (Table 1). LVWI determined after 4 days of treatment was significantly lower in rats receiving SODB at 4 and 40U or enalapril when compared to untreated SHR (2.45 ± 0.08, 2.54 ± 0.06 and 2.36 ± 0.06 versus 2.89 ± 0.03 mg HW. g1 BW, respectively). After 28 days of observation, LV mass index was further lowered in enalapril-treated SHR, while no additional reduction was detected with the two doses of SODB (Table 1). As presented in Figure 2, cardiomyocyte diameter was significantly reduced by SODB from the fourth day. Water intake and excretion were stable in the untreated SHR while a parallel rise was observed for both parameters in the three treated groups (data not shown). Thus, water balance was comparable in all groups (Table 1). Oxidative status As shown in Figure 3 the two doses of SODB induced a significant increase in the cardiac quantity of total SOD and GPx after 28 days of treatment (by 30–40% compared to untreated SHR), while enalapril had no effect on these expressions. CAT expression was not modulated in any group. As shown in Figure 4, DHE intensity was significantly reduced by the two doses of SODB with an average reduction of 13% compared to untreated SHR. No significant difference was detected in cardiac superoxide anion production in SODB- and enalapril-treated SHR. Inflammatory status None of the treatments affected cardiac concentration of the nuclear factor NF-kB. Moreover, no difference was observed in left ventricular IL-6, IL-1b or TNF-a concentrations in any group (Table 2). Plasma concentration of IL-6, IL-1b or TNF-a was similar in untreated- and SODB-treated SHR. Enalapril administration was associated with a reduction in the three markers and significance was only achieved for TNF-a (Table 2).
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Figure 2. Influence of short- (4 days) and long- (28 days) term treatment with SODB or enalapril on cardiomyocyte size. Upper panel: Cardiomyocyte staining with hematoxylin-eosin at the end of observation period (bar ¼ 25 mm). Lower panel: Cardiomyocyte size determined on at least 40 transverse sections per heart in 6 and 12 rats per group for the short- and long-term treatment, respectively. *p50.05 compared to untreated SHR for 4 and 28 days of treatment.
Discussion The present study demonstrates that genetically hypertensive rats treated with SODB have a reduced cardiac mass. The lowering of cardiac mass was equated with the LVWI and confirmed very clearly by direct measurement of cardiomyocytes diameter on tissue slices. The effect of SODB was observed similarly with the two doses used in the study (4 or 40 U SOD/day). The cardiac effect was detected since the first measurement, i.e. after 4 days of treatment, and was maintained after the 4 weeks of SODB administration. Among the various parameters which could participate in the reduction of cardiac mass is the lowering of preload. During the 28 days of treatment, neither water intake nor urine volume was modified by SODB, while both parameters increased to the same extent during enalapril administration. Although no direct measurement of blood volume or venous return was performed in the present experiment, the lack of change in water handling does not favor a major involvement of preload reduction in the beneficial cardiac influence of the melon SOD.
A reduction of afterload may also partly explain the cardiac effect of SODB administration. Yet, the decrease in arterial pressure was slight as measured indirectly in conscious rats or directly recorded in anesthetized animals. As observed on cardiac mass, the decrease in arterial blood pressure was similar with the two doses of SODB. In addition, the peak effect of SODB on blood pressure was achieved after four days of treatment and remained stable throughout. The slight antihypertensive influence of SODB might be related to its ACE inhibitor activity previously reported in vitro using hippuryl-L-histidyl-L-leucine as the substrate (Carillon et al., 2012). In order to investigate such an effect, the pressure response to exogenous AngI administration was evaluated in rats treated for 4 days only, when the blood pressure and cardiac mass lowering effect of SODB were already maximal. In contrast to that observed with enalapril, the peak response was not affected by SODB treatment even at the highest dose. Although we cannot exclude a very early effect of SODB particularly in cardiac tissue, the inhibition of ACE does not seem to play a major role in the effect of SODB in vivo. Interestingly,
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DOI: 10.3109/09637486.2014.893286
Melon SOD reduces cardiac hypertrophy
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Figure 3. Influence of long-term administration of SODB or enalapril on cardiac antioxidant defense expression. (A) Cardiac total SOD expression (Cu/Zn-SOD and Mn-SOD). (B) Cardiac GPx expression. (C) Cardiac CAT expression. Quantification was made after standardization within membranes by expressing the density of each band of interest relative to that of GAPDH in the same lane. Results are then expressed as relative changes from untreated SHR band intensity. *p50.05 compared to untreated SHR.
blood pressure reduction was weaker with SODB than enalapril (5 versus 20%, approximately), while cardiac hypertrophy was reduced to the same extent with the two compounds. A reduction in arterial pressure is not necessarily required for the beneficial effect of treatments on cardiac hypertrophy. A non-antihypertensive dose of enalapril in SHR (Piotrkowski et al., 2009) or ramipril in aortic banding hypertension (Linz et al., 1992) prevent cardiac tissue remodeling. A non-angiotensin-related compound such as resveratrol also prevents the development of cardiac hypertrophy in the SHR independently of blood pressure reduction (Thandapilly et al., 2010). Conversely, hydralazine administration has no effect on cardiac mass despite a marked reduction of blood pressure in the SHR (Gupta et al., 2005). Altogether, these findings indicate that the beneficial effect on cardiac hypertrophy cannot be entirely explained by reduction in pressure or volume overload, and suggest that SODB has an additional influence independently of the circulating renin–angiotensin system. In the current study, none of the two doses of SODB reduced NF-kB or the pro-inflammatory cytokines concentrations in cardiac tissue. In addition, SODB did not influence circulating cytokines. Although a very early effect cannot be ruled out, our results do not favor a major involvement of an anti-inflammatory effect in the beneficial influence of SODB. Besides hemodynamic changes or anti-inflammatory influence, reduction in the oxidative status appears to be an important
mechanism of the antihypertrophic effect of SODB. No significant change in chemiluminescence intensity using lucigenin probe was detected. Although our present observation must be confirmed on unfixed frozen tissue, a slight reduction in the staining by DHE of formalin-fixed, paraffin-embedded cardiac tissue was observed in rats treated by SODB. If we assume that lucigenin (in the presence of KCN) provides a chemiluminescent substrate to monitor superoxide production by NADPH oxidase and that DHE is a reliable marker of O2 intracellular presence, it is suggested that O2 scavenging rather than formation was mainly involved in the antihypertrophic effect of SODB in our setting. Interestingly, SODB administration was associated with an increased expression of endogenous antioxidant defenses (SOD and GPx). Potent induction of cellular antioxidant enzymes by grape seed polyphenols (Du et al., 2007) was previously reported in cultured rat cardiac H9C2 cells. Zhou et al. (2012) have also shown that osthol, a natural coumarin compound, could induce endogenous SOD and GPx in hypertrophied heart of rats. In recent studies, we have shown that SODB supplementation for 28 days could increase endogenous antioxidant enzymes (SOD, GPx and CAT) in the liver and adipose tissue of obese hamsters (Carillon et al., 2013b,c), and in the liver of healthy rats (Carillon et al., 2013a). Even if SODB was coated in order to reach intestinal mucosa, SOD protein is probably not absorbed. The question arises of how an exogenous melon SOD administration, could induce endogenous antioxidant defense?
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Figure 4. Influence of 28 days of treatment with SODB or enalapril on dihydroethidine staining. Upper panel: Example of DHE staining of left ventricle slices at the end of observation period ( 100). Lower panel: DHE intensity size determined on at least 10 transverse sections per heart. *p50.05 compared to untreated SHR.
Table 2. Oxidative and inflammatory status of the heart of SHR after the 28 days of SODB administration.
Untreated
Enalapril
SODB 4U
SODB 40 U
Cardiac O2 detection by Lucigenin 9420 ± 1523 6302 ± 398 8249 ± 592 6209 ± 821 (RLU/mg prot) Cardiac concentration (ng/mg prot) IL-1b 5.9 ± 0.7 IL-6 11.8 ± 3.2 TNF-a 7.5 ± 1.3 NF-kB 50.6 ± 8.6
6.1 ± 0.9 14.3 ± 2.4 8.7 ± 1.3 38.5 ± 4.6
6.7 ± 1.2 6.8 ± 0.6 11.7 ± 4.0 12.0 ± 3.1 8.8 ± 1.5 9.8 ± 1.0 50.3 ± 11.2 36.7 ± 8.2
Plasma concentration (pg/mL) IL-1b 76 ± 8 IL-6 408 ± 79 TNF-a 112 ± 6
57 ± 6 335 ± 49 76 ± 9*
102 ± 34 464 ± 33 81 ± 14
109 ± 26 452 ± 50 91 ± 17
Values are means ± SE. RLU: relative light units; IL-1b: interleukin1beta; IL-6: interleukin-6; TNF-a: tumor necrosis factor-alpha; NF-kB: nuclear factor-kappaB. *p50.05 compared to untreated SHR.
Other exogenous SODs seem to act by inducing endogenous antioxidant defense, as reviewed by Carillon et al. (2013d), and some hypothesis have been proposed even if the precise mechanism is still unknown. Indeed, the nuclear factor-E2-related factor (Nrf2)/antioxidant response element (ARE) pathway (Li et al., 2012), may be involved in the induction of endogenous antioxidant enzymes by SOD. The higher amount of endogenous antioxidant enzymes in cardiac tissue and probably the subsequent reduction of cardiac oxidative stress could explain the anti-hypertrophic and antihypertensive effects of SODB. An influence of antioxidant defense on cardiac morphology was previously suggested by the reduction of both Mn-SOD and CuZn-SOD in the hypertrophied heart of aged SHR (Ito et al., 1995). In mice, SOD deficiency was associated with a more marked LVH after myocardial infarction (van Deel et al., 2008). SOD and GPx deficiency was also observed in the blood of patients with permanent essential hypertension (Iarema et al., 1992). On the other hand, treatment with tempol, a SOD mimetic, had antihypertensive effect on SHR (Peixoto et al., 2009) and attenuated cardiac hypertrophy in GLUT4-knockout insulin-resistant mice (Ritchie et al., 2007). Consistent with the latter study using tempol
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DOI: 10.3109/09637486.2014.893286
(Ritchie et al., 2007), SODB induced a reduction in cardiac hypertrophy even at a dose (4U) insufficient to lower cardiac superoxide anion production. Since a concomitant stimulation of SOD and GPx was observed, the low dose of SODB probably also acted through the blunting of oxidative stress by subsequent endogenous antioxidant enzymes. Heart growth associated with pathological situation implicates a large number of signaling pathways (see Heineke & Molkentin, 2006 for review), where oxidative stress and redox signaling had probably a substantial role (review by Seddon et al., 2007). Which are the precise molecular targets of the cardiovascular effects of SODB remain to be investigated in future dedicated experiments. The induction of endogenous antioxidant defense after SODB administration could explain why high dose (40U) and long-term supplementation (28 days) did not exert more benefits. Our results suggest that a supplementation of 4 days with 4U of SODB is sufficient to induce a stock of endogenous antioxidant enzymes. In addition, other doses (10 and 80U) have been assessed in the same conditions, and no more beneficial effects have been observed (data not shown). Moreover, the induction of several antioxidant enzymes avoids imbalance, which could be involved in some diseases such as Down syndrome (Muchova et al., 2001; Perluigi & Butterfield, 2012). Indeed, GPx removes H2O2 produced after dismutation of O2 by SOD.
Conclusions In summary, we have shown that SODB has a beneficial effect on cardiac hypertrophy in a model of genetic hypertension. Such an effect was related to a reduction of oxidative stress through stimulation of endogenous antioxidant defense in cardiac tissue rather than hemodynamic or anti-inflammatory influence. These findings suggest that SODB could play an important role in cardiac protection in essential hypertension. Its beneficial influence as a dietary supplementation during conventional antihypertensive therapy has to be investigated. In addition, the effect of SODB on other tissues and/or different pathologies that involve oxidative stress deserves further investigations.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. Julie Carillon was supported by a ‘‘CIFRE grant’’ (Convention Industrielle de Formation par la REcherche, n 0417/2010) from Bionov (Avignon, France) and the French ‘‘Association Nationale de la Recherche et de la Technologie’’. Dominique Lacan is Bionov R&D director.
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