- Email: [email protected]
Heme oxygenase-1 alleviates vascular complications associated with metabolic syndrome: Effect on endothelial dependent relaxation and NO production
Chemico-Biological Interactions 223 (2014) 109–115
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Heme oxygenase-1 alleviates vascular complications associated with metabolic syndrome: Effect on endothelial dependent relaxation and NO production Hany M. El-Bassossy a,b,⇑, Nadia Hassan b, Mohamed N.M. Zakaria b a b
Department of Pharmacology, Faculty of Pharmacy, King Abdulaziz University, Saudi Arabia Department of Pharmacology, Faculty of Pharmacy, Zagazig University, Egypt
a r t i c l e
i n f o
Article history: Received 6 August 2014 Received in revised form 17 September 2014 Accepted 22 September 2014 Available online 28 September 2014 Keywords: Metabolic syndrome Aorta Hypertension Haem oxygenase-1
a b s t r a c t Protective effect of Heme oxygenase-1 (HO-1) induction from hypertension was previously reported in a diabetic animal model. Here, the effect of HO-1 induction on vascular complications associated with metabolic syndrome (MetS) was investigated. MetS was induced in rats by fructose drinking for 12 weeks while HO-1 was induced by hemin or curcumin administration in the last 6 weeks. Then, aortic HO-1 protein expression was assessed, blood pressure (BP) was recorded and serum levels of glucose and insulin were measured. Concentration response curves for phenylephrine (PE), KCl, and acetylcholine (ACh) were obtained in thoracic aortic cross sections. Aortic reactive oxygen species (ROS) and nitric oxide (NO) generation were also studied. Both hemin and curcumin significantly inhibited the elevated systolic and diastolic BP seen in MetS animals. While not affected by MetS, HO-1 expression was significantly increased by hemin and curcumin treatment. HO-1 induction did not affect the exaggerated vasoconstriction response to KCl and PE. However, HO-1 induction prevented the impaired relaxation and NO generation in aorta isolated from MetS animals. In addition, the HO inhibitor, tin protoporphyrin, abolished the hemin protective effect on relaxation and NO generation. HO-1 induction prevented the elevated hyperinsulinemia associated with MetS. Furthermore, HO-1 induction inhibited ROS production in aorta isolated from MetS animals. In conclusion, Heme oxygenase-1 alleviates vascular complications associated in MetS through maintaining endothelial-dependent relaxation and NO generation in addition to improving insulin sensitivity. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Metabolic syndrome (MetS) is a group of metabolic risk factors concurrently occurs; includes insulin resistance, hypertension, and obesity. These factors increase dramatically the risk of cardiovascular complications [1]. Some studies suggest a strong association between insulin resistance and endothelial dysfunction [2]. The intake of fructose is raised with an increased consumption of soft drinks and many beverages containing high fructose in recent Abbreviations: ANOVA, analysis of variance; eNOS, endothelial nitric oxide synthase; HO-1, Haem oxygenase-1; MetS, metabolic syndrome; NO, nitric oxide; PE, phenylephrine; ROS, reactive oxygen species; SnPP, tin protoporphyrin; STZ, streptozotocin. ⇑ Corresponding author at: Department of Pharmacology, Faculty of Pharmacy, King Abdulaziz University, P.O. Box 80260, Jeddah 21589, Saudi Arabia. Mobile: +966 568751075. E-mail addresses: [email protected], [email protected] (H.M. El-Bassossy). http://dx.doi.org/10.1016/j.cbi.2014.09.014 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
years. Thus, fructose has been implicated as the useful tool to induce MetS in animals [3]. Recent findings support that the increased consumption of fructose may be an important contributor to the metabolic syndrome, typically resulting in hyperinsulinemia, insulin resistance and hypertension [4]. Heme oxygenase (HO) enzyme is one of the important homeostatic mechanisms in the body that has been reported to modulate cellular responses following tissue injury [5]. HO has three isoforms; HO-1 which is the most important isoform that is induced following tissue injury, HO-2 which is the constitutive isoform and HO-3 that is not expressed in human [6]. HO-1 catalyzes heme degradation that result in release of iron (which increases ferritin level), biliverdin (which is converted to bilirubin) and carbon monoxide (CO). Ferritin and bilirubin processes potent antioxidant properties while CO induced antioxidant genes [6] and has vasodilator activities [6,7]. Protective effect of HO-1 induction against development of hypertension was previously reported in a diabetic animal model [8]. Among the HO-1 inducers, hemin is a standard
110
H.M. El-Bassossy et al. / Chemico-Biological Interactions 223 (2014) 109–115
inducer that is used in both in vitro and in vivo [9,10]. Curcumin is a natural polyphenolic compound that has been described as HO-1 inducer in vivo [8]. Both hemin and curcumin induced HO-1 expression and HO activity while tin protoporphyrin significantly inhibited HO activity in pulmonary arteries in previous work [11]. Therefore, the current study was designed to determine the potentially protective effect of HO-1 induction against vascular complications associated with MetS and illustrate the mechanism of the possible protection.
2. Materials and methods 2.1. Animals Male Wistar rats (strain code: 003; Zagazig University, Zagazig, Egypt) of 120–130 g body weight were housed at 3–4 rats per cage in clear polypropylene cages and kept on under stable environmental conditions and equal light–dark cycle. Rats with initial body weight 120–130 g were found to be the most sensitive age to MetS induction by fructose in preliminary experiments. Rats had free access to the commercially available rodent pellet diet and purified water. The animal care and handling were according to the institutional guidelines that are in accordance with European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes. Experimental protocol was approved by Zagazig Ethical Committee for Animal Handling.
2.3. Blood pressure measurement Blood pressure (BP) was measured indirectly in a conscious and slightly restrained rat by the tail cuff method as described previously [14]. Rats were conditioned to the warming chamber and the restraint for 10–20 min/day for 3 days before measurements. BP measurements were carried out between 7:00 and 12:00 AM by the same investigator. After a stabilization period (5–10 min) in the warming chamber (35 °C), 10 repetitions of the automated inflation–deflation cycle were performed. The mean of 6 readings within a 5–10 mmHg range was considered as the BP. 2.4. HO-1 immunofluorescence Immunofluorescence staining of HO-1 protein expression in rat paraffin embedded aorta sections was carried out according to the method detailed in previous work [8]. Non-specific binding was blocked by incubating the sections with fluorescence-enhanced blocking buffer (Molecular Probes, Paisley, UK) for 30 min. The method uses rabbit polyclonal primary antibody (dilution 1:200, StressGen, Ann Arbor, MI, USA) to detect the HO-1 in aortic sections followed by Alexa fluor conjugated secondary antibody (dilution 1:1000, Molecular probes, UK). Images were obtained by LEICA DM500 fluorescence microscope with fluorescent cubic set at ex 480/30 and em 525/40 (Leica Microsystems, Wetzlar, Germany). Images were acquired with identical acquisition parameters. Fluorescence quantification was carried out by Image J software. Staining aortae sections with the secondary antibody only did not show any specific staining. 2.5. Serum analysis
2.2. Study protocol In ex vivo study, animals were randomly divided into six experimental groups (8 animals each); control, metabolic syndrome (MetS), hemin-treated metabolic syndrome (MetS-Hemin), curcumin-treated metabolic syndrome (MetS-curcumin). MetS was induced by adding fructose (10%) to the drinking water. MetS was confirmed by a stable hyperinsulinemia (6–8 lIU/ml) after 6 weeks of fructose drinking when MetS rats are divided between groups [12]. Rats were then received hemin (10 mg kg1) or curcumin (5 mg kg1). hemin and curcumin were firstly dissolved in 0.5 N NaOH in dark and quickly diluted 200-fold with phosphate buffer saline and injected to rats intraperitoneally for 6 weeks. Control and resistant groups receive 0.5 N NaOH and diluted 200-fold with phosphate buffer saline as a vehicle. The dose and method of preparation of hemin and curcumin were chosen as employed in previous work on HO-1 induction [8]. Twelve hours after the last injection, blood pressure was measured. Blood was collected from the retro-orbital plexus under light ether anesthesia during blood collection (2–3 min) followed by termination of animal life by cervical dislocation. The collected blood was then centrifuged (3000g, 4 °C,20 min) to separate serum that was analyzed for glucose and insulin. Then, the thoracic aorta was carefully excised and placed in cold Krebs–Henseleit buffer (KHB) with the following composition; NaCl 118.1, KCl 4.69, KH2PO4 1.2, NaHCO3 25.0, glucose 11.7, MgSO4 0.5 and CaCl2 2.5 mM. The aorta was then cleaned of fat and connective tissue and cut into 4 rings (3 mm length). For every animal, one aortic cross section was suspended in an isolated tissue bath system for studying vascular reactivity while the other four rings of aorta were used to study ACh-induced NO generation, reactive oxygen species (ROS) generation and HO-1 immunofluorescence. In some experiments, a fifth aortic cross section from diabetic-hemin animals were incubated with SnPP (20 lM, 1 h) and suspended in an tissue bath for studying contractility responses.
Serum glucose was determined by glucose meter (Bionime GmbH, Berneck, Switzerland) using noble metal electrode strips. Insulin level in serum was determined by sandwich ELISA kit that uses microplate strips coated with monoclonal mouse antibody against rat insulin (Millipore, Cairo, Egypt). The insulin resistance index (HOMA-IR) was estimated using the following equation: HOMA-IR = glucose concentration (mmol/l) insulin (lU/l)/22.5 [13]. 2.6. Vascular reactivity Vascular reactivity of isolated thoracic aortae was performed using the isolated artery techniques as full described in previous publications [15–17]. For studying the aortic contractile responsiveness, the increases in tension to cumulative additions of phenylephrine (PE, 109 to 105 M) or KCl (10 to 100 mM) was recorded. In order to study the vasodilator responsiveness of aorta, rings were first pre-contracted with submaximal concentrations of PE. The cumulative concentrations of acetylcholine (ACh, 109 to 105 M) were then added to the organ bath and the response was recorded. The submaximal concentration of PE was chosen to give similar pre-contraction in all studied groups. 2.7. Intracellular reactive oxygen species (ROS) generation Intracellular generation of ROS in aorta was determined according to methods described in details in previous publication [8]. Briefly, frozen aorta was homogenized in sucrose/tween 80 solution to solubilized lipids then centrifuged. The supernatant was incubated with 2,7-dichlorofluresceindiacetate (DCF-DA) in buffer at 37 °C for 2 h. At the end of incubation, the formation of the fluorescent oxidized derivative DCF was measured using a Perkin ElmerÒ spectrophotofluorometer LS45 (PerkinElmer, Cairo, Egypt) with excitation set at 488 nm and emission set at 525 nm.
H.M. El-Bassossy et al. / Chemico-Biological Interactions 223 (2014) 109–115
111
2.8. ACh-induced NO generation The intracellular NO generation following ACh activation of isolated aorta was investigated according to the method described in details in previous publications [15,18]. Briefly, isolated aortic cross sections were loaded by the fluorescence probes 4-amino5-methylamino-20 ,70 -difluorofluorescein diacetate (DAF-FM) by incubation in buffer at 37 °C for 30 min. Then the aortic cross sections were opened longitudinally, placed in a specific chamber with the endothelial side up for fluorescence measurements using a LS45 fluorescence spectrophotometer (PerkinElmerÒ, Cairo, Egypt) with remote fiber optic with excitation set at 485 nm and emission set at 515 nm. 2.9. Drugs and chemicals The following drugs and chemicals were used: Fructose (El-Nasr Chemical Co., Cairo, Egypt), Ach, PE, Hemin, SN-PP and Curcumin (Sigma–Aldrich, Munich, Germany). ACh and PE were dissolved in cold Krebs–Henseleit buffer. 2.10. Statistical analysis Values are expressed as mean ± SEM. The agonist maximum response (Emax) was calculated from concentration–response curve by non-linear regression analysis of individual curves using computer based fitting program (Prism 5, Graphpad, CA, USA). Statistical analysis was performed by the analysis of variance (ANOVA) followed by Newman-Keuls’ post hoc test. 3. Results 3.1. Blood pressure Fructose-feeding was associated with significant elevations in both systolic blood pressure and diastolic blood pressure (p < 0.001) compared to control group, while no significant change in pulse pressure. On the other hand, both HO-1 inducers, hemin and curcumin led to a significant decrease in both systolic and diastolic blood pressure (all at p < 0.001) compared to fructose-fed group, while no significant change in pulse pressure (Fig. 1). 3.2. HO-1 immunofluorescence HO-1 immunofluorescence staining was found in all layers of aorta cross sections. Aortae isolated from fructose (10% in drinking water)-fed animals did not show any significant change in HO-1 expression compared with control. While both hemin and curcumin showed a significant increase in the expression of HO-1 (p < 0.001) compared with the fructose-fed group (Fig. 2).
Fig. 1. Effect of daily intraperitoneally administration of hemin (10 mg kg1) or curcumin (5 mg kg1) on the systolic (a), diastolic (b) and pulse (c) blood pressure (BP) in fructose-induced metabolic syndrome (MetS, 10% in drinking water, for 12 weeks). ⁄⁄⁄p < 0.001, compared with the corresponding control group values; ### p < 0.001 compared with the corresponding MetS group values; by One Way ANOVA and Newman Keuls post hoc test.
by the decrease in serum insulin and insulin resistance index compared with fructose-fed animals (all at p < 0.001). However, neither fructose feeding nor HO-1 induction affected the blood glucose level (Table 1). 3.4. Vascular reactivity
3.3. Metabolic syndrome parameters Caloric intake was 541 kcal/day/kg in the control group and 618 kcal/day/kg in the MetS groups. There was no significant difference in the fluid consumption between the groups. Daily administration of fructose (10%) to the drinking water for 12 weeks led to a significant increase in body weight (p < 0.001) compared to control group. However, HO-1 induction by hemin or curcumin did not significantly affect developed over weight in fructose-fed animals. Fructose administration led also to a marked insulin resistance as indicated by the significant elevation on serum insulin (p < 0.001) and the insulin resistance index (p < 0.01) compared with control. HO-1 induction by hemin or curcumin completely inhibited the developed insulin resistance in fructose-fed animals as indicated
Aorta isolated from MetS animals showed exaggerated vasoconstriction responses to KCl and PE, reflected by a significant increase in apparent Emax (both at p < 0.001) compared to control group. HO-1 induction by hemin or curcumin did not have any significant effect on aorta contraction to KCl while curcumin treatment was associated with a significant decrease in aorta contraction in response to PE compared with MetS group (p < 0.001, Fig. 3a and b). On the other hand, MetS resulted in a large decrease in aorta relaxation to ACh, reflected by a significant decrease in apparent Emax (p < 0.05) compared with the control. HO-1 induction by hemin and curcumin administration led to a significant increase in relaxation responsiveness to ACh, reflected by a significant increases in the apparent Emax (p < 0.01, p < 0.05, respectively)
112
H.M. El-Bassossy et al. / Chemico-Biological Interactions 223 (2014) 109–115
Fig. 2. Effect of daily intraperitoneally administration of hemin (10 mg kg1) or curcumin (5 mg kg1) on the thoracic aorta HO-1 expression in fructose-induced metabolic syndrome (MetS, 10% in drinking water, for 12 weeks). Symbols indicate mean ± SEM for N = 8 animals; ⁄⁄⁄p < 0.001, compared with the corresponding control group values; by One Way ANOVA and Newman Keuls post hoc test. Micrographs at the top are representative fluorescence images of aorta cross sections (endothelial side on left) immunofluorescence stained by heme-oxygenase-1 antibodies followed by Alexa fluor conjugated secondary antibodies.
compared with the MetS group. Incubation of aorta isolated from hemin-treated MetS animals for 1 h with HO-1 inhibitor (SnPP) prevented the protective effect of hemin on aorta relaxation (p < 0.01, Fig. 4a). 3.5. ACh-induced NO generation Fructose-induced MetS significantly inhibited ACh stimulated NO release compared with control animals as reflected by a significant decrease in apparent Emax (p < 0.01). While HO-1 induction by hemin and curcumin administration prevented the attenuated ACh-induced NO release seen in MetS reflected by a significant increase in apparent Emax (p < 0.001, p < 0.05, respectively) compared with MetS animals. Incubation of aorta isolated from hemin-treated MetS animals for 1 h with HO-1 inhibitor (SnPP) blocked the protective effect of hemin, reflected by a significant decrease in apparent Emax (p < 0.05, Fig. 4b). 3.6. ROS generation Aortae isolated from MetS animals were characterized by increased ROS generation (p < 0.05) compared to control. HO-1 induction by hemin or curcumin significantly inhibited the exaggerated ROS generation seen in aorta isolated from MetS animals (p < 0.01 and p < 0.05, respectively; Fig. 5). 4. Discussion The current study is the first to report on the potential protective effect of HO-1 induction against the hypertension and vascular complications associated with metabolic syndrome (MetS). HO-1
Fig. 3. Effect of daily intraperitoneally administration of hemin (10 mg kg1) or curcumin (5 mg kg1) on the isolated aorta responsiveness to KCl (a) and PE (b) in fructose-induced metabolic syndrome (MetS, 10% in drinking water, for 12 weeks). ⁄⁄⁄ p < 0.001, compared with the corresponding group values by One Way ANOVA and Newman Keuls post hoc test.
induction markedly inhibited the hypertension in fructose-induced MetS in rats. The following results can explain the possible mechanism of alleviation of hypertension associated with MetS by HO-1: (i) HO-1 induction ameliorated the MetS associated AChinduced relaxations, (ii) the exaggerated ROS generation and impaired NO observed in MetS were abrogated by HO-1 induction, (iii) HO-1 induction prevented the development of MetS. These findings provide convincing evidence that HO-1 enzyme offsets the hypertension and vascular complications accompanying MetS through endothelial dependent relaxation-NO signaling protection. It is reasoned that HO-1 induction would mitigate or at least minimize the hypertension and vascular complications associated with MetS. The validity of this assumption was ascertained in this work as will be discussed below. In this study including fructose
Table 1 Effect of daily intraperitoneally administration of hemin (10 mg kg1) or curcumin (5 mg kg1) on the body weight, blood glucose, serum insulin, insulin resistance index (IR), systolic and diastolic BP and aortic reactive oxygen species (ROS) generation in fructose-induced metabolic syndrome (MetS, 10% in drinking water, for 12 weeks). Treatment
Body weight (g)
Glucose (mg dl1)
Insulin (lg l1)
IR index (units)
Systolic BP (mmHg)
Diastolic BP (mmHg)
ROS (fluorescent units)
Control MetS MetS–hemin MetS–curcumin
255.8 ± 7.2 379.9 ± 13.6⁄⁄⁄ 375.2 ± 12.3 350.1 ± 22.8
115.7 ± 5.9 111.7 ± 6.6 113.7 ± 2.7 114.2 ± 4.1
10.8 ± 0.4 16.3 ± 1.0⁄⁄⁄ 5.2 ± 1.4### 4.2 ± 0.8###
2.8 ± 0.1 4.3 ± 0.4⁄⁄ 1.3 ± 0.4### 1.4 ± 0.3###
102.7 ± 1.1 124.7 ± 3.1⁄⁄⁄ 112.7 ± 1.4### 108.2 ± 1.9###
83.0 ± 0.9 99.7 ± 1.9⁄⁄⁄ 87.8 ± 0.7### 85.1 ± 0.9###
358.0 ± 76.2 615.8 ± 68.4⁄ 220.9 ± 56.5## 286.1 ± 68.8#
Values are expressed as the mean ± S.E of mean; N = 8 animals; ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001, compared with the corresponding control group values; #p < 0.05, ##p < 0.01, ### p < 0.001 compared with the corresponding MetS group values; by One Way ANOVA and Newman Keuls post hoc test.
H.M. El-Bassossy et al. / Chemico-Biological Interactions 223 (2014) 109–115
Fig. 4. Effect of daily intraperitoneally administration of hemin (10 mg kg1) or curcumin (5 mg kg1) on the isolated aorta responsiveness to ACh (a) and AChstimulated NO generation in fructose-induced metabolic syndrome (MetS, 10% in drinking water, for 12 weeks). ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001, compared with the corresponding group values by One Way ANOVA and Newman Keuls post hoc test.
Fig. 5. Effect of daily intraperitoneally administration of hemin (10 mg kg1) or curcumin (5 mg kg1) on the aortic reactive oxygen species (ROS) generation in fructose-induced metabolic syndrome (MetS, 10% in drinking water, for 12 weeks). ⁄ p < 0.05, compared with the corresponding group values; #p < 0.05, ##p < 0.01 compared with the corresponding MetS group values by One Way ANOVA and Newman Keuls post hoc test.
(10%) in drinking water developed significant hyperinsulinemia, elevated insulin resistance index in 6 weeks. This is considered the most reliable model of insulin resistance [19]. The insulin resistant animals in this study were characterized by elevations in both
113
systolic and diastolic blood pressure which is in consistence with previous results [14]. The increased systolic (afterload) BP may be secondary to decreased myocardial contractility [20] while the elevated diastolic (preload) BP is attributed to the impaired vascular reactivity observed in MetS animals. HO-1 enzyme was previously induced in aorta by hemin (10 mg kg1) or curcumin (5 mg kg1) in vivo. HO-1 induction by hemin or curcumin protected against the progression of hypertension induced by MetS as reflected by the significant reduction in both systolic and diastolic blood pressure compared with MetS animals. Previously, induced HO-1 expression was found to regulate hypertension in young spontaneously hypertensive rats [21,22]. The protective effect of HO-1 enzyme observed in the study seems to be due to both direct vascular protective effect exerted by the enzyme and indirect effect through inhibition of the insulin resistance as seen in this study. The study focused on the impairment in vascular reactivity because of its importance in the development of hypertension [23] and in insulin resistance-evoked hypertension in particular [24]. Exaggerated contraction of isolated aorta to PE and KCl and decreased relaxation to ACh was found in insulin resistant animals. These findings are supported by previous reports, which showed that MetS is accompanied with increased response to different vasoconstrictors [25,26] and impaired endothelium-dependent relaxation [16,27,28]. As increased production of superoxide anions, mediated mainly by hyperinsulinemia associated with MetS, leads to reduced synthesis of nitric oxide from the endothelium and/or inactivation of nitric oxide released from the endothelium, and ultimately to impaired vascular responses [29–31]. Constitutively generated ROS from endothelial cells, in addition to exerting contractions per se, augment the response of various contractile agents in endothelium intact rat aortic cross sections [32]. In the present work, HO-1 induction by both hemin and curcumin prevented the impaired relaxation to ACh in aorta isolated from animals with MetS without affecting the response to KCl. Functional previous studies using aortic cross sections from rabbit showed that HO-1 induction enhanced endothelium-dependent relaxation [33]. In addition, HO inhibition by SnPP, which is known to be a selective inhibitor to HO-1 [34] blocked the protective effect of hemin against the MetS-induced impaired endotheliumdependent relaxation. These data confirm that the observed protection was due to the induction of HO-1 and was not a consequence of a direct effect of hemin on the endothelium. On the other hand, The protective effect of curcumin against exaggerated response to PE seen in this study is most likely not mediated by HO-1 as hemin did not has such protective effect. The mechanism(s) by which HO-1 induction protected against endothelium-dependent relaxation impairment could be mediated, by restoration of NO production. In the present study, both HO-1 inducers prevented the impaired NO generation following ACh-stimulated in MetS aorta. This is in harmony with previous literature where, up-regulation of HO-1 expression increased eNOS expression [35]. HO-1 per se and heme degradation products possess antioxidant and anti-inflammatory activities which are required for vascular NO preservation; hence HO-1 expression may increase NO bioavailability [36]. There are three possible scenarios for preserving vascular NO via HO-1; (1) modulate eNOS expression and activity, (2) prevent inactivation of vascular NO, and (3) compensate for the loss of vascular NO [36]. The effect on reactive oxygen species (ROS) formation is an important mechanism by which HO-1 induction protected against the impairment in endothelium-dependent relaxation. Clinical and experimental studies have provided evidence that endogenous or exogenous ROS can modulate the vascular tone and perhaps act as a mediator for signal transduction in endothelial cells [37]. ROS mediate a decrease in NO bioavailability and endothelial
114
H.M. El-Bassossy et al. / Chemico-Biological Interactions 223 (2014) 109–115
dysfunction, with secondary oxidized and nitrated by-products of these reactions contributing to the pathogenesis of numerous vascular diseases [38]. The MetS induced by fructose was accompanied with elevation in ROS which is inconsistence with previous studies [39,40]. HO-1 induction by hemin and curcumin down-regulated the generation of reactive oxygen species. This is in consistence with previous study in which HO-1 induction attenuates the cisplatin-induced apoptosis of auditory cells via down-regulation of reactive oxygen species generation [41]. Induction of HO-1 is commonly considered to enhance the antioxidant activity of cells and to provide in vivo protection against conditions associated with oxidative stress [42]. Conversely, cells overexpressing HO-1 have been reported to be more resistant to oxidant-induced toxicity than the corresponding control cells [43]. The HO-1 products, biliverdin and bilirubin, are recognized as potent antioxidants manufactured by the body, because they can directly scavenge ROS and reduce ROS production through undefined mechanisms [44]. Furthermore, CO was found to protect against superoxideanions-induced hepatocytes apoptosis [45] and inhibit hyperoxic apoptosis by inhibiting cellular ROS production [46]. In conclusion, HO-1 induction by hemin or curcumin alleviates hypertension and vascular complications associated with MetS by direct and indirect protective mechanisms. The direct protective mechanism is through maintenance of endothelial-dependent relaxation and NO generation, while the indirect mechanism is through inhibition of the MetS. 5. Limitation The differences between animal and human in the vasculature, the endocrine system and the molecular pathways in addition to the relatively low number of evaluated animals (8 per group), make the obtained results are rudimentary for the human risk assessment. However, the current study are not focused on this. In addition, the Ethical committee recommended 6–8 animals in each group in order to follow the reduction rule in animal research. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version. References [1] E. Nisoli, E. Clementi, M.O. Carruba, S. Moncada, Defective mitochondrial biogenesis: a hallmark of the high cardiovascular risk in the metabolic syndrome?, Circ Res. 100 (2007) 795–806. [2] H.O. Steinberg, H. Chaker, R. Leaming, A. Johnson, G. Brechtel, A.D. Baron, Obesity/insulin resistance is associated with endothelial dysfunction. Implications for the syndrome of insulin resistance, J. Clin. Invest. 97 (1996) 2601–2610. [3] J.H. Hsu, Y.C. Wu, I.M. Liu, J.T. Cheng, Dioscorea as the principal herb of DieHuang-Wan, a widely used herbal mixture in China, for improvement of insulin resistance in fructose-rich chow-fed rats, J. Ethnopharmacol. 112 (2007) 577–584. [4] W. Suwannaphet, A. Meeprom, S. Yibchok-Anun, S. Adisakwattana, Preventive effect of grape seed extract against high-fructose diet-induced insulin resistance and oxidative stress in rats, Food Chem. Toxicol. 48 (2010) 1853– 1857. [5] M. Pope, Stress proteins and the immune response, Immunopharmacology 48 (2000) 299–302. [6] N.G. Abraham, A. Kappas, Heme oxygenase and the cardiovascular-renal system, Free Radic. Biol. Med. 39 (2005) 1–25. [7] C.W. Leffler, A. Nasjletti, C. Yu, R.A. Johnson, A.L. Fedinec, N. Walker, Carbon monoxide and cerebral microvascular tone in newborn pigs, Am. J. Physiol. Heart Circ. Physiol. 276 (1999) H1641–H1646.
[8] N. Hassan, H.M. El-Bassossy, M.N.M. Zakaria, Heme oxygenase-1 induction protects against hypertension associated with diabetes: effect on exaggerated vascular contractility, Naunyn Schmiedebergs Arch. Pharmacol. 386 (2013) 217–226. [9] J. Alam, E. Killeen, P. Gong, R. Naquin, B. Hu, D. Stewart, J.R. Ingelfinger, K.A. Nath, Heme activates the heme oxygenase-1 gene in renal epithelial cells by stabilizing Nrf2, Am. J. Physiol. Renal Physiol. 284 (2003) F743–F752. [10] S.K.R. Kanakiriya, A.J. Croatt, J.J. Haggard, J.R. Ingelfinger, S.S. Tang, J. Alam, K.A. Nath, Heme: a novel inducer of MCP-1 through HO-dependent and HOindependent mechanisms, Am. J. Physiol. Renal Physiol. 284 (2003) F546– F554. [11] H.M. El-Bassossy, N.N. El-Maraghy, H.M. El-Fayoumi, M.L. Watson, Haem oxygenase-1 induction protects against tumour necrosis factor a impairment of endothelial-dependent relaxation in rat isolated pulmonary artery, Br. J. Pharmacol. 158 (2009) 1527–1535. [12] H. El-Bassossy, N. Dsokey, A. Fahmy, Characterization of vascular complications in experimental model of fructose induced metabolic syndrome, Toxicol. Mech. Methods (2014) 1–8. [13] D.R. Matthews, J.P. Hosker, A.S. Rudenski, B.A. Naylor, D.F. Treacher, R.C. Turner, Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man, Diabetologia 28 (1985) 412–419. [14] H.M. El-Bassossy, A. Fahmy, D. Badawy, Cinnamaldehyde protects from the hypertension associated with diabetes, Food Chem. Toxicol. 49 (2011) 3007– 3012. [15] H.M. El-Bassossy, M.A. El-Moselhy, M.F. Mahmoud, Pentoxifylline alleviates vascular impairment in insulin resistance via TNF-a inhibition, Naunyn Schmiedebergs Arch. Pharmacol. 384 (2011) 277–285. [16] H.M. El-Bassossy, R. El-Fawal, A. Fahmy, M.L. Watson, Arginase inhibition alleviates hypertension in the metabolic syndrome, Br. J. Pharmacol. 169 (2013) 693–703. [17] M.F. Mahmoud, N.A. Hassan, H.M. El Bassossy, A. Fahmy, Quercetin protects against diabetes-induced exaggerated vasoconstriction in rats: effect on low grade inflammation, PLoS One 8 (5) (2013) e63784. [18] H.M. El-Bassossy, R. El-Fawal, A. Fahmy, Arginase inhibition alleviates hypertension associated with diabetes: effect on endothelial dependent relaxation and NO production, Vascul. Pharmacol. 57 (2012) 194–200. [19] S. Dai, J.H. McNeill, Fructose-induced hypertension in rats is concentrationand duration-dependent, J. Pharmacol. Toxicol. Methods 33 (1995) 101–107. [20] R. Danielsen, J.E. Nordrehaug, E. Lien, H. Vik-Mo, Subclinical left ventricular abnormalities in young subjects with long-term type 1 diabetes mellitus detected by digitized m-mode echocardiography, Am. J. Cardiol. 60 (1987) 143–146. [21] B. Escalante, D. Sacerdoti, M.M. Davidian, M. Laniado-Schwartzman, J.C. McGiff, Chronic treatment with tin normalizes blood pressure in spontaneously hypertensive rats, Hypertension 17 (1991) 776–779. [22] J.F. Ndisang, W. Zhao, R. Wang, Selective regulation of blood pressure by heme oxygenase-1 in hypertension, Hypertension 40 (2002) 315–321. [23] S.M. Mansour, A.K. Bahgat, A.S. El-Khatib, M.T. Khayyal, Ginkgo biloba extract (EGb 761) normalizes hypertension in 2K, 1C hypertensive rats: role of antioxidant mechanisms, ACE inhibiting activity and improvement of endothelial dysfunction, Phytomedcine 18 (2011) 641–647. [24] Ansarullah, B. Bharucha, V. Patel, A.V. Ramachandran, Oreocnide integrifolia (Gaud.) Miq leaf water extract improves metabolic alterations in high fructose fed insulin resistant and hypertensive rats, Eur. J. Integr. Med. 2 (2010) 79–87. [25] D. Badawy, H.M. El-Bassossy, A. Fahmy, A. Azhar, Aldose reductase inhibitors zopolrestat and ferulic acid alleviate hypertension associated with diabetes: effect on vascular reactivity, Can. J. Physiol. Pharmacol. 91 (2013) 101–107. [26] S.N. Iyer, M.J. Katovich, Vascular reactivity to phenylephrine and angiotensin II in hypertensive rats associated with insulin resistance, Clin. Exp. Hypertens. 18 (1996) 227–242. [27] L. Lind, Endothelium-dependent vasodilation, insulin resistance and the metabolic syndrome in an elderly cohort: The Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study, Atherosclerosis 196 (2008) 795–802. [28] S. Verma, S. Bhanot, L. Yao, J.H. McNeill, Defective endothelium-dependent relaxation in fructose-hypertensive rats, Am. J. Hypertens. 9 (1996) 370–376. [29] G. Kojda, D. Harrison, Interactions between NO and reactive oxygen species: pathophysiological importance in atherosclerosis, hypertension, diabetes and heart failure, Cardiovasc. Res. 43 (1999) 652–671. [30] D.W. Laight, M.J. Carrier, E.E. Anggard, Antioxidants, diabetes and endothelial dysfunction, Cardiovasc. Res. 47 (2000) 457–464. [31] K. Mellor, R.H. Ritchie, G. Meredith, O.L. Woodman, M.J. Morris, L.M.D. Delbridge, High-fructose diet elevates myocardial superoxide generation in mice in the absence of cardiac hypertrophy, Nutrition 26 (2010) 842–848. [32] P. Srivastava, L.G. Hegde, G.K. Patnaik, M. Dikshit, Role of endothelial-derived reactive oxygen species and nitric oxide in norepinephrine-induced rat aortic ring contractions, Pharmacol. Res. 38 (1998) 265–274. [33] B.J. Wu, K. Kathir, P.K. Witting, K. Beck, K. Choy, C. Li, K.D. Croft, T.A. Mori, D. Tanous, M.R. Adams, A.K. Lau, R. Stocker, Antioxidants protect from atherosclerosis by a heme oxygenase-1 pathway that is independent of free radical scavenging, J. Exp. Med. 203 (2006) 1117–1127. [34] G.A. Visner, F. Lu, H. Zhou, J. Liu, K. Kazemfar, A. Agarwal, Rapamycin induces heme oxygenase-1 in human pulmonary vascular cells: implications in the antiproliferative response to rapamycin, Circulation 107 (2003) 911–916.
H.M. El-Bassossy et al. / Chemico-Biological Interactions 223 (2014) 109–115 [35] M. Ahmad, S. Turkseven, C.J. Mingone, S.A. Gupte, M.S. Wolin, N.G. Abraham, Heme oxygenase-1 gene expression increases vascular relaxation and decreases inducible nitric oxide synthase in diabetic rats, Cell. Mol. Biol. (Noisy-le-grand) 51 (2005) 371–376. [36] H.O. Pae, Y. Son, N.H. Kim, H.J. Jeong, K.C. Chang, H.T. Chung, Role of heme oxygenase in preserving vascular bioactive NO, Nitric Oxide 23 (2010) 251– 257. [37] T. Finkel, Oxygen radicals and signaling, Curr. Opin. Cell Biol. 10 (1998) 248– 253. [38] N.K.H. Khoo, V. Rudolph, M.P. Cole, F. Golin-Bisello, F.J. Schopfer, S.R. Woodcock, C. Batthyany, B.A. Freeman, Activation of vascular endothelial nitric oxide synthase and heme oxygenase-1 expression by electrophilic nitrofatty acids, Free Radic. Biol. Med. 48 (2010) 230–239. [39] S. Delbosc, E. Paizanis, R. Magous, C. Araiz, T.o. Dimo, J.P. Cristol, G. Cros, J. Azay, Involvement of oxidative stress and NADPH oxidase activation in the development of cardiovascular complications in a model of insulin resistance, the fructose-fed rat, Atherosclerosis 179 (2005) 43–49. [40] K.M. Mellor, J.R. Bell, M.J. Young, R.H. Ritchie, L.M.D. Delbridge, Myocardial autophagy activation and suppressed survival signaling is associated with insulin resistance in fructose-fed mice, J. Mol. Cell. Cardiol. 50 (2011) 1035– 1043.
115
[41] H.J. Kim, H.S. So, J.H. Lee, J.H. Lee, C. Park, S.Y. Park, Y.H. Kim, M.J. Youn, S.J. Kim, S.Y. Chung, K.M. Lee, R. Park, Heme oxygenase-1 attenuates the cisplatininduced apoptosis of auditory cells via down-regulation of reactive oxygen species generation, Free Radic. Biol. Med. 40 (2006) 1810–1819. [42] R. Stocker, M.A. Perrella, Heme oxygenase-1: a novel drug target for atherosclerotic diseases?, Circulation 114 (2006) 2178–2189 [43] R. Hori, M. Kashiba, T. Toma, A. Yachie, N. Goda, N. Makino, A. Soejima, T. Nagasawa, K. Nakabayashi, M. Suematsu, Gene transfection of H25A mutant heme oxygenase-1 protects cells against hydroperoxide-induced cytotoxicity, J. Biol. Chem. 277 (2002) 10712–10718. [44] R. Stocker, Antioxidant activities of bile pigments, Antioxid. Redox Signal. 6 (2004) 841–849. [45] L. Conde de la Rosa, T.E. Vrenken, R.A. Hannivoort, M. Buist-Homan, R. Havinga, D.J. Slebos, H.F. Kauffman, K.N. Faber, P.L.M. Jansen, H. Moshage, Carbon monoxide blocks oxidative stress-induced hepatocyte apoptosis via inhibition of the p54 JNK isoform, Free Radic. Biol. Med. 44 (2008) 1323– 1333. [46] X. Wang, Y. Wang, H.P. Kim, K. Nakahira, S.W. Ryter, A.M.K. Choi, Carbon monoxide protects against hyperoxia-induced endothelial cell apoptosis by inhibiting reactive oxygen species formation, J. Biol. Chem. 282 (2007) 1718– 1726.
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Heme oxygenase-1 alleviates vascular complications associated with metabolic syndrome: Effect on endothelial dependent relaxation and NO production Hany M. El-Bassossy a,b,⇑, Nadia Hassan b, Mohamed N.M. Zakaria b a b
Department of Pharmacology, Faculty of Pharmacy, King Abdulaziz University, Saudi Arabia Department of Pharmacology, Faculty of Pharmacy, Zagazig University, Egypt
a r t i c l e
i n f o
Article history: Received 6 August 2014 Received in revised form 17 September 2014 Accepted 22 September 2014 Available online 28 September 2014 Keywords: Metabolic syndrome Aorta Hypertension Haem oxygenase-1
a b s t r a c t Protective effect of Heme oxygenase-1 (HO-1) induction from hypertension was previously reported in a diabetic animal model. Here, the effect of HO-1 induction on vascular complications associated with metabolic syndrome (MetS) was investigated. MetS was induced in rats by fructose drinking for 12 weeks while HO-1 was induced by hemin or curcumin administration in the last 6 weeks. Then, aortic HO-1 protein expression was assessed, blood pressure (BP) was recorded and serum levels of glucose and insulin were measured. Concentration response curves for phenylephrine (PE), KCl, and acetylcholine (ACh) were obtained in thoracic aortic cross sections. Aortic reactive oxygen species (ROS) and nitric oxide (NO) generation were also studied. Both hemin and curcumin significantly inhibited the elevated systolic and diastolic BP seen in MetS animals. While not affected by MetS, HO-1 expression was significantly increased by hemin and curcumin treatment. HO-1 induction did not affect the exaggerated vasoconstriction response to KCl and PE. However, HO-1 induction prevented the impaired relaxation and NO generation in aorta isolated from MetS animals. In addition, the HO inhibitor, tin protoporphyrin, abolished the hemin protective effect on relaxation and NO generation. HO-1 induction prevented the elevated hyperinsulinemia associated with MetS. Furthermore, HO-1 induction inhibited ROS production in aorta isolated from MetS animals. In conclusion, Heme oxygenase-1 alleviates vascular complications associated in MetS through maintaining endothelial-dependent relaxation and NO generation in addition to improving insulin sensitivity. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Metabolic syndrome (MetS) is a group of metabolic risk factors concurrently occurs; includes insulin resistance, hypertension, and obesity. These factors increase dramatically the risk of cardiovascular complications [1]. Some studies suggest a strong association between insulin resistance and endothelial dysfunction [2]. The intake of fructose is raised with an increased consumption of soft drinks and many beverages containing high fructose in recent Abbreviations: ANOVA, analysis of variance; eNOS, endothelial nitric oxide synthase; HO-1, Haem oxygenase-1; MetS, metabolic syndrome; NO, nitric oxide; PE, phenylephrine; ROS, reactive oxygen species; SnPP, tin protoporphyrin; STZ, streptozotocin. ⇑ Corresponding author at: Department of Pharmacology, Faculty of Pharmacy, King Abdulaziz University, P.O. Box 80260, Jeddah 21589, Saudi Arabia. Mobile: +966 568751075. E-mail addresses: [email protected], [email protected] (H.M. El-Bassossy). http://dx.doi.org/10.1016/j.cbi.2014.09.014 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
years. Thus, fructose has been implicated as the useful tool to induce MetS in animals [3]. Recent findings support that the increased consumption of fructose may be an important contributor to the metabolic syndrome, typically resulting in hyperinsulinemia, insulin resistance and hypertension [4]. Heme oxygenase (HO) enzyme is one of the important homeostatic mechanisms in the body that has been reported to modulate cellular responses following tissue injury [5]. HO has three isoforms; HO-1 which is the most important isoform that is induced following tissue injury, HO-2 which is the constitutive isoform and HO-3 that is not expressed in human [6]. HO-1 catalyzes heme degradation that result in release of iron (which increases ferritin level), biliverdin (which is converted to bilirubin) and carbon monoxide (CO). Ferritin and bilirubin processes potent antioxidant properties while CO induced antioxidant genes [6] and has vasodilator activities [6,7]. Protective effect of HO-1 induction against development of hypertension was previously reported in a diabetic animal model [8]. Among the HO-1 inducers, hemin is a standard
110
H.M. El-Bassossy et al. / Chemico-Biological Interactions 223 (2014) 109–115
inducer that is used in both in vitro and in vivo [9,10]. Curcumin is a natural polyphenolic compound that has been described as HO-1 inducer in vivo [8]. Both hemin and curcumin induced HO-1 expression and HO activity while tin protoporphyrin significantly inhibited HO activity in pulmonary arteries in previous work [11]. Therefore, the current study was designed to determine the potentially protective effect of HO-1 induction against vascular complications associated with MetS and illustrate the mechanism of the possible protection.
2. Materials and methods 2.1. Animals Male Wistar rats (strain code: 003; Zagazig University, Zagazig, Egypt) of 120–130 g body weight were housed at 3–4 rats per cage in clear polypropylene cages and kept on under stable environmental conditions and equal light–dark cycle. Rats with initial body weight 120–130 g were found to be the most sensitive age to MetS induction by fructose in preliminary experiments. Rats had free access to the commercially available rodent pellet diet and purified water. The animal care and handling were according to the institutional guidelines that are in accordance with European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes. Experimental protocol was approved by Zagazig Ethical Committee for Animal Handling.
2.3. Blood pressure measurement Blood pressure (BP) was measured indirectly in a conscious and slightly restrained rat by the tail cuff method as described previously [14]. Rats were conditioned to the warming chamber and the restraint for 10–20 min/day for 3 days before measurements. BP measurements were carried out between 7:00 and 12:00 AM by the same investigator. After a stabilization period (5–10 min) in the warming chamber (35 °C), 10 repetitions of the automated inflation–deflation cycle were performed. The mean of 6 readings within a 5–10 mmHg range was considered as the BP. 2.4. HO-1 immunofluorescence Immunofluorescence staining of HO-1 protein expression in rat paraffin embedded aorta sections was carried out according to the method detailed in previous work [8]. Non-specific binding was blocked by incubating the sections with fluorescence-enhanced blocking buffer (Molecular Probes, Paisley, UK) for 30 min. The method uses rabbit polyclonal primary antibody (dilution 1:200, StressGen, Ann Arbor, MI, USA) to detect the HO-1 in aortic sections followed by Alexa fluor conjugated secondary antibody (dilution 1:1000, Molecular probes, UK). Images were obtained by LEICA DM500 fluorescence microscope with fluorescent cubic set at ex 480/30 and em 525/40 (Leica Microsystems, Wetzlar, Germany). Images were acquired with identical acquisition parameters. Fluorescence quantification was carried out by Image J software. Staining aortae sections with the secondary antibody only did not show any specific staining. 2.5. Serum analysis
2.2. Study protocol In ex vivo study, animals were randomly divided into six experimental groups (8 animals each); control, metabolic syndrome (MetS), hemin-treated metabolic syndrome (MetS-Hemin), curcumin-treated metabolic syndrome (MetS-curcumin). MetS was induced by adding fructose (10%) to the drinking water. MetS was confirmed by a stable hyperinsulinemia (6–8 lIU/ml) after 6 weeks of fructose drinking when MetS rats are divided between groups [12]. Rats were then received hemin (10 mg kg1) or curcumin (5 mg kg1). hemin and curcumin were firstly dissolved in 0.5 N NaOH in dark and quickly diluted 200-fold with phosphate buffer saline and injected to rats intraperitoneally for 6 weeks. Control and resistant groups receive 0.5 N NaOH and diluted 200-fold with phosphate buffer saline as a vehicle. The dose and method of preparation of hemin and curcumin were chosen as employed in previous work on HO-1 induction [8]. Twelve hours after the last injection, blood pressure was measured. Blood was collected from the retro-orbital plexus under light ether anesthesia during blood collection (2–3 min) followed by termination of animal life by cervical dislocation. The collected blood was then centrifuged (3000g, 4 °C,20 min) to separate serum that was analyzed for glucose and insulin. Then, the thoracic aorta was carefully excised and placed in cold Krebs–Henseleit buffer (KHB) with the following composition; NaCl 118.1, KCl 4.69, KH2PO4 1.2, NaHCO3 25.0, glucose 11.7, MgSO4 0.5 and CaCl2 2.5 mM. The aorta was then cleaned of fat and connective tissue and cut into 4 rings (3 mm length). For every animal, one aortic cross section was suspended in an isolated tissue bath system for studying vascular reactivity while the other four rings of aorta were used to study ACh-induced NO generation, reactive oxygen species (ROS) generation and HO-1 immunofluorescence. In some experiments, a fifth aortic cross section from diabetic-hemin animals were incubated with SnPP (20 lM, 1 h) and suspended in an tissue bath for studying contractility responses.
Serum glucose was determined by glucose meter (Bionime GmbH, Berneck, Switzerland) using noble metal electrode strips. Insulin level in serum was determined by sandwich ELISA kit that uses microplate strips coated with monoclonal mouse antibody against rat insulin (Millipore, Cairo, Egypt). The insulin resistance index (HOMA-IR) was estimated using the following equation: HOMA-IR = glucose concentration (mmol/l) insulin (lU/l)/22.5 [13]. 2.6. Vascular reactivity Vascular reactivity of isolated thoracic aortae was performed using the isolated artery techniques as full described in previous publications [15–17]. For studying the aortic contractile responsiveness, the increases in tension to cumulative additions of phenylephrine (PE, 109 to 105 M) or KCl (10 to 100 mM) was recorded. In order to study the vasodilator responsiveness of aorta, rings were first pre-contracted with submaximal concentrations of PE. The cumulative concentrations of acetylcholine (ACh, 109 to 105 M) were then added to the organ bath and the response was recorded. The submaximal concentration of PE was chosen to give similar pre-contraction in all studied groups. 2.7. Intracellular reactive oxygen species (ROS) generation Intracellular generation of ROS in aorta was determined according to methods described in details in previous publication [8]. Briefly, frozen aorta was homogenized in sucrose/tween 80 solution to solubilized lipids then centrifuged. The supernatant was incubated with 2,7-dichlorofluresceindiacetate (DCF-DA) in buffer at 37 °C for 2 h. At the end of incubation, the formation of the fluorescent oxidized derivative DCF was measured using a Perkin ElmerÒ spectrophotofluorometer LS45 (PerkinElmer, Cairo, Egypt) with excitation set at 488 nm and emission set at 525 nm.
H.M. El-Bassossy et al. / Chemico-Biological Interactions 223 (2014) 109–115
111
2.8. ACh-induced NO generation The intracellular NO generation following ACh activation of isolated aorta was investigated according to the method described in details in previous publications [15,18]. Briefly, isolated aortic cross sections were loaded by the fluorescence probes 4-amino5-methylamino-20 ,70 -difluorofluorescein diacetate (DAF-FM) by incubation in buffer at 37 °C for 30 min. Then the aortic cross sections were opened longitudinally, placed in a specific chamber with the endothelial side up for fluorescence measurements using a LS45 fluorescence spectrophotometer (PerkinElmerÒ, Cairo, Egypt) with remote fiber optic with excitation set at 485 nm and emission set at 515 nm. 2.9. Drugs and chemicals The following drugs and chemicals were used: Fructose (El-Nasr Chemical Co., Cairo, Egypt), Ach, PE, Hemin, SN-PP and Curcumin (Sigma–Aldrich, Munich, Germany). ACh and PE were dissolved in cold Krebs–Henseleit buffer. 2.10. Statistical analysis Values are expressed as mean ± SEM. The agonist maximum response (Emax) was calculated from concentration–response curve by non-linear regression analysis of individual curves using computer based fitting program (Prism 5, Graphpad, CA, USA). Statistical analysis was performed by the analysis of variance (ANOVA) followed by Newman-Keuls’ post hoc test. 3. Results 3.1. Blood pressure Fructose-feeding was associated with significant elevations in both systolic blood pressure and diastolic blood pressure (p < 0.001) compared to control group, while no significant change in pulse pressure. On the other hand, both HO-1 inducers, hemin and curcumin led to a significant decrease in both systolic and diastolic blood pressure (all at p < 0.001) compared to fructose-fed group, while no significant change in pulse pressure (Fig. 1). 3.2. HO-1 immunofluorescence HO-1 immunofluorescence staining was found in all layers of aorta cross sections. Aortae isolated from fructose (10% in drinking water)-fed animals did not show any significant change in HO-1 expression compared with control. While both hemin and curcumin showed a significant increase in the expression of HO-1 (p < 0.001) compared with the fructose-fed group (Fig. 2).
Fig. 1. Effect of daily intraperitoneally administration of hemin (10 mg kg1) or curcumin (5 mg kg1) on the systolic (a), diastolic (b) and pulse (c) blood pressure (BP) in fructose-induced metabolic syndrome (MetS, 10% in drinking water, for 12 weeks). ⁄⁄⁄p < 0.001, compared with the corresponding control group values; ### p < 0.001 compared with the corresponding MetS group values; by One Way ANOVA and Newman Keuls post hoc test.
by the decrease in serum insulin and insulin resistance index compared with fructose-fed animals (all at p < 0.001). However, neither fructose feeding nor HO-1 induction affected the blood glucose level (Table 1). 3.4. Vascular reactivity
3.3. Metabolic syndrome parameters Caloric intake was 541 kcal/day/kg in the control group and 618 kcal/day/kg in the MetS groups. There was no significant difference in the fluid consumption between the groups. Daily administration of fructose (10%) to the drinking water for 12 weeks led to a significant increase in body weight (p < 0.001) compared to control group. However, HO-1 induction by hemin or curcumin did not significantly affect developed over weight in fructose-fed animals. Fructose administration led also to a marked insulin resistance as indicated by the significant elevation on serum insulin (p < 0.001) and the insulin resistance index (p < 0.01) compared with control. HO-1 induction by hemin or curcumin completely inhibited the developed insulin resistance in fructose-fed animals as indicated
Aorta isolated from MetS animals showed exaggerated vasoconstriction responses to KCl and PE, reflected by a significant increase in apparent Emax (both at p < 0.001) compared to control group. HO-1 induction by hemin or curcumin did not have any significant effect on aorta contraction to KCl while curcumin treatment was associated with a significant decrease in aorta contraction in response to PE compared with MetS group (p < 0.001, Fig. 3a and b). On the other hand, MetS resulted in a large decrease in aorta relaxation to ACh, reflected by a significant decrease in apparent Emax (p < 0.05) compared with the control. HO-1 induction by hemin and curcumin administration led to a significant increase in relaxation responsiveness to ACh, reflected by a significant increases in the apparent Emax (p < 0.01, p < 0.05, respectively)
112
H.M. El-Bassossy et al. / Chemico-Biological Interactions 223 (2014) 109–115
Fig. 2. Effect of daily intraperitoneally administration of hemin (10 mg kg1) or curcumin (5 mg kg1) on the thoracic aorta HO-1 expression in fructose-induced metabolic syndrome (MetS, 10% in drinking water, for 12 weeks). Symbols indicate mean ± SEM for N = 8 animals; ⁄⁄⁄p < 0.001, compared with the corresponding control group values; by One Way ANOVA and Newman Keuls post hoc test. Micrographs at the top are representative fluorescence images of aorta cross sections (endothelial side on left) immunofluorescence stained by heme-oxygenase-1 antibodies followed by Alexa fluor conjugated secondary antibodies.
compared with the MetS group. Incubation of aorta isolated from hemin-treated MetS animals for 1 h with HO-1 inhibitor (SnPP) prevented the protective effect of hemin on aorta relaxation (p < 0.01, Fig. 4a). 3.5. ACh-induced NO generation Fructose-induced MetS significantly inhibited ACh stimulated NO release compared with control animals as reflected by a significant decrease in apparent Emax (p < 0.01). While HO-1 induction by hemin and curcumin administration prevented the attenuated ACh-induced NO release seen in MetS reflected by a significant increase in apparent Emax (p < 0.001, p < 0.05, respectively) compared with MetS animals. Incubation of aorta isolated from hemin-treated MetS animals for 1 h with HO-1 inhibitor (SnPP) blocked the protective effect of hemin, reflected by a significant decrease in apparent Emax (p < 0.05, Fig. 4b). 3.6. ROS generation Aortae isolated from MetS animals were characterized by increased ROS generation (p < 0.05) compared to control. HO-1 induction by hemin or curcumin significantly inhibited the exaggerated ROS generation seen in aorta isolated from MetS animals (p < 0.01 and p < 0.05, respectively; Fig. 5). 4. Discussion The current study is the first to report on the potential protective effect of HO-1 induction against the hypertension and vascular complications associated with metabolic syndrome (MetS). HO-1
Fig. 3. Effect of daily intraperitoneally administration of hemin (10 mg kg1) or curcumin (5 mg kg1) on the isolated aorta responsiveness to KCl (a) and PE (b) in fructose-induced metabolic syndrome (MetS, 10% in drinking water, for 12 weeks). ⁄⁄⁄ p < 0.001, compared with the corresponding group values by One Way ANOVA and Newman Keuls post hoc test.
induction markedly inhibited the hypertension in fructose-induced MetS in rats. The following results can explain the possible mechanism of alleviation of hypertension associated with MetS by HO-1: (i) HO-1 induction ameliorated the MetS associated AChinduced relaxations, (ii) the exaggerated ROS generation and impaired NO observed in MetS were abrogated by HO-1 induction, (iii) HO-1 induction prevented the development of MetS. These findings provide convincing evidence that HO-1 enzyme offsets the hypertension and vascular complications accompanying MetS through endothelial dependent relaxation-NO signaling protection. It is reasoned that HO-1 induction would mitigate or at least minimize the hypertension and vascular complications associated with MetS. The validity of this assumption was ascertained in this work as will be discussed below. In this study including fructose
Table 1 Effect of daily intraperitoneally administration of hemin (10 mg kg1) or curcumin (5 mg kg1) on the body weight, blood glucose, serum insulin, insulin resistance index (IR), systolic and diastolic BP and aortic reactive oxygen species (ROS) generation in fructose-induced metabolic syndrome (MetS, 10% in drinking water, for 12 weeks). Treatment
Body weight (g)
Glucose (mg dl1)
Insulin (lg l1)
IR index (units)
Systolic BP (mmHg)
Diastolic BP (mmHg)
ROS (fluorescent units)
Control MetS MetS–hemin MetS–curcumin
255.8 ± 7.2 379.9 ± 13.6⁄⁄⁄ 375.2 ± 12.3 350.1 ± 22.8
115.7 ± 5.9 111.7 ± 6.6 113.7 ± 2.7 114.2 ± 4.1
10.8 ± 0.4 16.3 ± 1.0⁄⁄⁄ 5.2 ± 1.4### 4.2 ± 0.8###
2.8 ± 0.1 4.3 ± 0.4⁄⁄ 1.3 ± 0.4### 1.4 ± 0.3###
102.7 ± 1.1 124.7 ± 3.1⁄⁄⁄ 112.7 ± 1.4### 108.2 ± 1.9###
83.0 ± 0.9 99.7 ± 1.9⁄⁄⁄ 87.8 ± 0.7### 85.1 ± 0.9###
358.0 ± 76.2 615.8 ± 68.4⁄ 220.9 ± 56.5## 286.1 ± 68.8#
Values are expressed as the mean ± S.E of mean; N = 8 animals; ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001, compared with the corresponding control group values; #p < 0.05, ##p < 0.01, ### p < 0.001 compared with the corresponding MetS group values; by One Way ANOVA and Newman Keuls post hoc test.
H.M. El-Bassossy et al. / Chemico-Biological Interactions 223 (2014) 109–115
Fig. 4. Effect of daily intraperitoneally administration of hemin (10 mg kg1) or curcumin (5 mg kg1) on the isolated aorta responsiveness to ACh (a) and AChstimulated NO generation in fructose-induced metabolic syndrome (MetS, 10% in drinking water, for 12 weeks). ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001, compared with the corresponding group values by One Way ANOVA and Newman Keuls post hoc test.
Fig. 5. Effect of daily intraperitoneally administration of hemin (10 mg kg1) or curcumin (5 mg kg1) on the aortic reactive oxygen species (ROS) generation in fructose-induced metabolic syndrome (MetS, 10% in drinking water, for 12 weeks). ⁄ p < 0.05, compared with the corresponding group values; #p < 0.05, ##p < 0.01 compared with the corresponding MetS group values by One Way ANOVA and Newman Keuls post hoc test.
(10%) in drinking water developed significant hyperinsulinemia, elevated insulin resistance index in 6 weeks. This is considered the most reliable model of insulin resistance [19]. The insulin resistant animals in this study were characterized by elevations in both
113
systolic and diastolic blood pressure which is in consistence with previous results [14]. The increased systolic (afterload) BP may be secondary to decreased myocardial contractility [20] while the elevated diastolic (preload) BP is attributed to the impaired vascular reactivity observed in MetS animals. HO-1 enzyme was previously induced in aorta by hemin (10 mg kg1) or curcumin (5 mg kg1) in vivo. HO-1 induction by hemin or curcumin protected against the progression of hypertension induced by MetS as reflected by the significant reduction in both systolic and diastolic blood pressure compared with MetS animals. Previously, induced HO-1 expression was found to regulate hypertension in young spontaneously hypertensive rats [21,22]. The protective effect of HO-1 enzyme observed in the study seems to be due to both direct vascular protective effect exerted by the enzyme and indirect effect through inhibition of the insulin resistance as seen in this study. The study focused on the impairment in vascular reactivity because of its importance in the development of hypertension [23] and in insulin resistance-evoked hypertension in particular [24]. Exaggerated contraction of isolated aorta to PE and KCl and decreased relaxation to ACh was found in insulin resistant animals. These findings are supported by previous reports, which showed that MetS is accompanied with increased response to different vasoconstrictors [25,26] and impaired endothelium-dependent relaxation [16,27,28]. As increased production of superoxide anions, mediated mainly by hyperinsulinemia associated with MetS, leads to reduced synthesis of nitric oxide from the endothelium and/or inactivation of nitric oxide released from the endothelium, and ultimately to impaired vascular responses [29–31]. Constitutively generated ROS from endothelial cells, in addition to exerting contractions per se, augment the response of various contractile agents in endothelium intact rat aortic cross sections [32]. In the present work, HO-1 induction by both hemin and curcumin prevented the impaired relaxation to ACh in aorta isolated from animals with MetS without affecting the response to KCl. Functional previous studies using aortic cross sections from rabbit showed that HO-1 induction enhanced endothelium-dependent relaxation [33]. In addition, HO inhibition by SnPP, which is known to be a selective inhibitor to HO-1 [34] blocked the protective effect of hemin against the MetS-induced impaired endotheliumdependent relaxation. These data confirm that the observed protection was due to the induction of HO-1 and was not a consequence of a direct effect of hemin on the endothelium. On the other hand, The protective effect of curcumin against exaggerated response to PE seen in this study is most likely not mediated by HO-1 as hemin did not has such protective effect. The mechanism(s) by which HO-1 induction protected against endothelium-dependent relaxation impairment could be mediated, by restoration of NO production. In the present study, both HO-1 inducers prevented the impaired NO generation following ACh-stimulated in MetS aorta. This is in harmony with previous literature where, up-regulation of HO-1 expression increased eNOS expression [35]. HO-1 per se and heme degradation products possess antioxidant and anti-inflammatory activities which are required for vascular NO preservation; hence HO-1 expression may increase NO bioavailability [36]. There are three possible scenarios for preserving vascular NO via HO-1; (1) modulate eNOS expression and activity, (2) prevent inactivation of vascular NO, and (3) compensate for the loss of vascular NO [36]. The effect on reactive oxygen species (ROS) formation is an important mechanism by which HO-1 induction protected against the impairment in endothelium-dependent relaxation. Clinical and experimental studies have provided evidence that endogenous or exogenous ROS can modulate the vascular tone and perhaps act as a mediator for signal transduction in endothelial cells [37]. ROS mediate a decrease in NO bioavailability and endothelial
114
H.M. El-Bassossy et al. / Chemico-Biological Interactions 223 (2014) 109–115
dysfunction, with secondary oxidized and nitrated by-products of these reactions contributing to the pathogenesis of numerous vascular diseases [38]. The MetS induced by fructose was accompanied with elevation in ROS which is inconsistence with previous studies [39,40]. HO-1 induction by hemin and curcumin down-regulated the generation of reactive oxygen species. This is in consistence with previous study in which HO-1 induction attenuates the cisplatin-induced apoptosis of auditory cells via down-regulation of reactive oxygen species generation [41]. Induction of HO-1 is commonly considered to enhance the antioxidant activity of cells and to provide in vivo protection against conditions associated with oxidative stress [42]. Conversely, cells overexpressing HO-1 have been reported to be more resistant to oxidant-induced toxicity than the corresponding control cells [43]. The HO-1 products, biliverdin and bilirubin, are recognized as potent antioxidants manufactured by the body, because they can directly scavenge ROS and reduce ROS production through undefined mechanisms [44]. Furthermore, CO was found to protect against superoxideanions-induced hepatocytes apoptosis [45] and inhibit hyperoxic apoptosis by inhibiting cellular ROS production [46]. In conclusion, HO-1 induction by hemin or curcumin alleviates hypertension and vascular complications associated with MetS by direct and indirect protective mechanisms. The direct protective mechanism is through maintenance of endothelial-dependent relaxation and NO generation, while the indirect mechanism is through inhibition of the MetS. 5. Limitation The differences between animal and human in the vasculature, the endocrine system and the molecular pathways in addition to the relatively low number of evaluated animals (8 per group), make the obtained results are rudimentary for the human risk assessment. However, the current study are not focused on this. In addition, the Ethical committee recommended 6–8 animals in each group in order to follow the reduction rule in animal research. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version. References [1] E. Nisoli, E. Clementi, M.O. Carruba, S. Moncada, Defective mitochondrial biogenesis: a hallmark of the high cardiovascular risk in the metabolic syndrome?, Circ Res. 100 (2007) 795–806. [2] H.O. Steinberg, H. Chaker, R. Leaming, A. Johnson, G. Brechtel, A.D. Baron, Obesity/insulin resistance is associated with endothelial dysfunction. Implications for the syndrome of insulin resistance, J. Clin. Invest. 97 (1996) 2601–2610. [3] J.H. Hsu, Y.C. Wu, I.M. Liu, J.T. Cheng, Dioscorea as the principal herb of DieHuang-Wan, a widely used herbal mixture in China, for improvement of insulin resistance in fructose-rich chow-fed rats, J. Ethnopharmacol. 112 (2007) 577–584. [4] W. Suwannaphet, A. Meeprom, S. Yibchok-Anun, S. Adisakwattana, Preventive effect of grape seed extract against high-fructose diet-induced insulin resistance and oxidative stress in rats, Food Chem. Toxicol. 48 (2010) 1853– 1857. [5] M. Pope, Stress proteins and the immune response, Immunopharmacology 48 (2000) 299–302. [6] N.G. Abraham, A. Kappas, Heme oxygenase and the cardiovascular-renal system, Free Radic. Biol. Med. 39 (2005) 1–25. [7] C.W. Leffler, A. Nasjletti, C. Yu, R.A. Johnson, A.L. Fedinec, N. Walker, Carbon monoxide and cerebral microvascular tone in newborn pigs, Am. J. Physiol. Heart Circ. Physiol. 276 (1999) H1641–H1646.
[8] N. Hassan, H.M. El-Bassossy, M.N.M. Zakaria, Heme oxygenase-1 induction protects against hypertension associated with diabetes: effect on exaggerated vascular contractility, Naunyn Schmiedebergs Arch. Pharmacol. 386 (2013) 217–226. [9] J. Alam, E. Killeen, P. Gong, R. Naquin, B. Hu, D. Stewart, J.R. Ingelfinger, K.A. Nath, Heme activates the heme oxygenase-1 gene in renal epithelial cells by stabilizing Nrf2, Am. J. Physiol. Renal Physiol. 284 (2003) F743–F752. [10] S.K.R. Kanakiriya, A.J. Croatt, J.J. Haggard, J.R. Ingelfinger, S.S. Tang, J. Alam, K.A. Nath, Heme: a novel inducer of MCP-1 through HO-dependent and HOindependent mechanisms, Am. J. Physiol. Renal Physiol. 284 (2003) F546– F554. [11] H.M. El-Bassossy, N.N. El-Maraghy, H.M. El-Fayoumi, M.L. Watson, Haem oxygenase-1 induction protects against tumour necrosis factor a impairment of endothelial-dependent relaxation in rat isolated pulmonary artery, Br. J. Pharmacol. 158 (2009) 1527–1535. [12] H. El-Bassossy, N. Dsokey, A. Fahmy, Characterization of vascular complications in experimental model of fructose induced metabolic syndrome, Toxicol. Mech. Methods (2014) 1–8. [13] D.R. Matthews, J.P. Hosker, A.S. Rudenski, B.A. Naylor, D.F. Treacher, R.C. Turner, Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man, Diabetologia 28 (1985) 412–419. [14] H.M. El-Bassossy, A. Fahmy, D. Badawy, Cinnamaldehyde protects from the hypertension associated with diabetes, Food Chem. Toxicol. 49 (2011) 3007– 3012. [15] H.M. El-Bassossy, M.A. El-Moselhy, M.F. Mahmoud, Pentoxifylline alleviates vascular impairment in insulin resistance via TNF-a inhibition, Naunyn Schmiedebergs Arch. Pharmacol. 384 (2011) 277–285. [16] H.M. El-Bassossy, R. El-Fawal, A. Fahmy, M.L. Watson, Arginase inhibition alleviates hypertension in the metabolic syndrome, Br. J. Pharmacol. 169 (2013) 693–703. [17] M.F. Mahmoud, N.A. Hassan, H.M. El Bassossy, A. Fahmy, Quercetin protects against diabetes-induced exaggerated vasoconstriction in rats: effect on low grade inflammation, PLoS One 8 (5) (2013) e63784. [18] H.M. El-Bassossy, R. El-Fawal, A. Fahmy, Arginase inhibition alleviates hypertension associated with diabetes: effect on endothelial dependent relaxation and NO production, Vascul. Pharmacol. 57 (2012) 194–200. [19] S. Dai, J.H. McNeill, Fructose-induced hypertension in rats is concentrationand duration-dependent, J. Pharmacol. Toxicol. Methods 33 (1995) 101–107. [20] R. Danielsen, J.E. Nordrehaug, E. Lien, H. Vik-Mo, Subclinical left ventricular abnormalities in young subjects with long-term type 1 diabetes mellitus detected by digitized m-mode echocardiography, Am. J. Cardiol. 60 (1987) 143–146. [21] B. Escalante, D. Sacerdoti, M.M. Davidian, M. Laniado-Schwartzman, J.C. McGiff, Chronic treatment with tin normalizes blood pressure in spontaneously hypertensive rats, Hypertension 17 (1991) 776–779. [22] J.F. Ndisang, W. Zhao, R. Wang, Selective regulation of blood pressure by heme oxygenase-1 in hypertension, Hypertension 40 (2002) 315–321. [23] S.M. Mansour, A.K. Bahgat, A.S. El-Khatib, M.T. Khayyal, Ginkgo biloba extract (EGb 761) normalizes hypertension in 2K, 1C hypertensive rats: role of antioxidant mechanisms, ACE inhibiting activity and improvement of endothelial dysfunction, Phytomedcine 18 (2011) 641–647. [24] Ansarullah, B. Bharucha, V. Patel, A.V. Ramachandran, Oreocnide integrifolia (Gaud.) Miq leaf water extract improves metabolic alterations in high fructose fed insulin resistant and hypertensive rats, Eur. J. Integr. Med. 2 (2010) 79–87. [25] D. Badawy, H.M. El-Bassossy, A. Fahmy, A. Azhar, Aldose reductase inhibitors zopolrestat and ferulic acid alleviate hypertension associated with diabetes: effect on vascular reactivity, Can. J. Physiol. Pharmacol. 91 (2013) 101–107. [26] S.N. Iyer, M.J. Katovich, Vascular reactivity to phenylephrine and angiotensin II in hypertensive rats associated with insulin resistance, Clin. Exp. Hypertens. 18 (1996) 227–242. [27] L. Lind, Endothelium-dependent vasodilation, insulin resistance and the metabolic syndrome in an elderly cohort: The Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study, Atherosclerosis 196 (2008) 795–802. [28] S. Verma, S. Bhanot, L. Yao, J.H. McNeill, Defective endothelium-dependent relaxation in fructose-hypertensive rats, Am. J. Hypertens. 9 (1996) 370–376. [29] G. Kojda, D. Harrison, Interactions between NO and reactive oxygen species: pathophysiological importance in atherosclerosis, hypertension, diabetes and heart failure, Cardiovasc. Res. 43 (1999) 652–671. [30] D.W. Laight, M.J. Carrier, E.E. Anggard, Antioxidants, diabetes and endothelial dysfunction, Cardiovasc. Res. 47 (2000) 457–464. [31] K. Mellor, R.H. Ritchie, G. Meredith, O.L. Woodman, M.J. Morris, L.M.D. Delbridge, High-fructose diet elevates myocardial superoxide generation in mice in the absence of cardiac hypertrophy, Nutrition 26 (2010) 842–848. [32] P. Srivastava, L.G. Hegde, G.K. Patnaik, M. Dikshit, Role of endothelial-derived reactive oxygen species and nitric oxide in norepinephrine-induced rat aortic ring contractions, Pharmacol. Res. 38 (1998) 265–274. [33] B.J. Wu, K. Kathir, P.K. Witting, K. Beck, K. Choy, C. Li, K.D. Croft, T.A. Mori, D. Tanous, M.R. Adams, A.K. Lau, R. Stocker, Antioxidants protect from atherosclerosis by a heme oxygenase-1 pathway that is independent of free radical scavenging, J. Exp. Med. 203 (2006) 1117–1127. [34] G.A. Visner, F. Lu, H. Zhou, J. Liu, K. Kazemfar, A. Agarwal, Rapamycin induces heme oxygenase-1 in human pulmonary vascular cells: implications in the antiproliferative response to rapamycin, Circulation 107 (2003) 911–916.
H.M. El-Bassossy et al. / Chemico-Biological Interactions 223 (2014) 109–115 [35] M. Ahmad, S. Turkseven, C.J. Mingone, S.A. Gupte, M.S. Wolin, N.G. Abraham, Heme oxygenase-1 gene expression increases vascular relaxation and decreases inducible nitric oxide synthase in diabetic rats, Cell. Mol. Biol. (Noisy-le-grand) 51 (2005) 371–376. [36] H.O. Pae, Y. Son, N.H. Kim, H.J. Jeong, K.C. Chang, H.T. Chung, Role of heme oxygenase in preserving vascular bioactive NO, Nitric Oxide 23 (2010) 251– 257. [37] T. Finkel, Oxygen radicals and signaling, Curr. Opin. Cell Biol. 10 (1998) 248– 253. [38] N.K.H. Khoo, V. Rudolph, M.P. Cole, F. Golin-Bisello, F.J. Schopfer, S.R. Woodcock, C. Batthyany, B.A. Freeman, Activation of vascular endothelial nitric oxide synthase and heme oxygenase-1 expression by electrophilic nitrofatty acids, Free Radic. Biol. Med. 48 (2010) 230–239. [39] S. Delbosc, E. Paizanis, R. Magous, C. Araiz, T.o. Dimo, J.P. Cristol, G. Cros, J. Azay, Involvement of oxidative stress and NADPH oxidase activation in the development of cardiovascular complications in a model of insulin resistance, the fructose-fed rat, Atherosclerosis 179 (2005) 43–49. [40] K.M. Mellor, J.R. Bell, M.J. Young, R.H. Ritchie, L.M.D. Delbridge, Myocardial autophagy activation and suppressed survival signaling is associated with insulin resistance in fructose-fed mice, J. Mol. Cell. Cardiol. 50 (2011) 1035– 1043.
115
[41] H.J. Kim, H.S. So, J.H. Lee, J.H. Lee, C. Park, S.Y. Park, Y.H. Kim, M.J. Youn, S.J. Kim, S.Y. Chung, K.M. Lee, R. Park, Heme oxygenase-1 attenuates the cisplatininduced apoptosis of auditory cells via down-regulation of reactive oxygen species generation, Free Radic. Biol. Med. 40 (2006) 1810–1819. [42] R. Stocker, M.A. Perrella, Heme oxygenase-1: a novel drug target for atherosclerotic diseases?, Circulation 114 (2006) 2178–2189 [43] R. Hori, M. Kashiba, T. Toma, A. Yachie, N. Goda, N. Makino, A. Soejima, T. Nagasawa, K. Nakabayashi, M. Suematsu, Gene transfection of H25A mutant heme oxygenase-1 protects cells against hydroperoxide-induced cytotoxicity, J. Biol. Chem. 277 (2002) 10712–10718. [44] R. Stocker, Antioxidant activities of bile pigments, Antioxid. Redox Signal. 6 (2004) 841–849. [45] L. Conde de la Rosa, T.E. Vrenken, R.A. Hannivoort, M. Buist-Homan, R. Havinga, D.J. Slebos, H.F. Kauffman, K.N. Faber, P.L.M. Jansen, H. Moshage, Carbon monoxide blocks oxidative stress-induced hepatocyte apoptosis via inhibition of the p54 JNK isoform, Free Radic. Biol. Med. 44 (2008) 1323– 1333. [46] X. Wang, Y. Wang, H.P. Kim, K. Nakahira, S.W. Ryter, A.M.K. Choi, Carbon monoxide protects against hyperoxia-induced endothelial cell apoptosis by inhibiting reactive oxygen species formation, J. Biol. Chem. 282 (2007) 1718– 1726.