Alarin alleviated cardiac fibrosis via attenuating oxidative stress in heart failure rats

The present study was to explore whether alarin could alleviate heart failure (HF) and attenuate cardia fibrosis via inhibiting oxidative stress. The fibrosis of cardiac fibroblasts (CFs) was induced by angiotensin (Ang) II. HF models were induced by ligation of the left anterior descending artery to cause ischemia myocardial infarction (MI) in Sprague–Dawley rats. Alarin (1.0 nM/kg/d) was administrated by intraperitoneal injection for 28 days. The decreases of left ventricular (LV) ejection fraction (EF), fractional shortening (FS), the maximum of the first differentiation of LV pressure (LV ± dp/dt_max) and LV systolic pressure (LVSP), and the increases of LV volume in systole (LVVS), LV volume in diastole (LVVD), LV end-systolic diameter (LVESD) and LV end-diastolic diameter (LVEDD) in MI rats were improved by alarin treatment. The increases in the expression levels of collagen I, collagen III, and transforming growth factor (TGF)-β were inhibited by alarin treatment in CFs and in the hearts of MI rats. The levels of NADPH oxidase (Nox) activity, superoxide anions and malondialdehyde (MDA) levels were increased, and the level of superoxide dismutase (SOD) activity was reduced in Ang II-treated CFs, which were reversed by alarin. Nox1 overexpression reversed the effects of alarin on attenuating the increases of collagen I, collagen III and TGF-β expression levels induced by Ang II in CFs. These results indicated that alarin improved HF and cardiac fibrosis via inhibiting oxidative stress in HF rats. Nox1 played important roles in the regulation of alarin effects on attenuating CFs fibrosis induced by Ang II.

     

In vivo TNF-alpha inhibition ameliorates cardiac mitochondrial dysfunction, oxidative stress, and apoptosis in experimental heart failure

Heart failure is associated with increased myocardial expression of TNF-alpha. However, the role of TNF-alpha in the development of heart failure is not fully understood. In the present study, we investigated the contribution of TNF-alpha to myocardial mitochondrial dysfunction, oxidative stress, and apoptosis in a unique dog model of heart failure characterized by an activation of all of these pathological processes. Male mongrel dogs were randomly assigned (n = 10 each) to 1) normal controls; 2) chronic pacing (250 beats/min for 4 wk) with concomitant administration of etanercept, a soluble p75 TNF receptor fusion protein, 0.5 mg/kg subcutaneously twice weekly; 3) chronic pacing with administration of saline vehicle. Mitochondrial function was assessed by left ventricular (LV) tissue mitochondrial respiratory enzyme activities. Oxidative stress was assessed with aldehyde levels, and apoptosis was quantified by photometric enzyme immunoassay for cytoplasmic histone-associated DNA fragments and terminal deoxynucleotide transferase-mediated nick-end labeling (TUNEL) assays. LV activity levels of mitochondrial respiratory chain enzyme complex III and V were reduced in the saline-treated dogs and restored either partially (complex III) or completely (complex V) in the etanercept-treated dogs. Aldehyde levels, DNA fragments, and TUNEL-positive cells were increased in the saline-treated dogs and normalized in etanercept-treated dogs. These changes were accompanied by an attenuation of LV dilatation and partial restoration of ejection fraction. Our data demonstrate that TNF-alpha contributes to progressive LV dysfunction in pacing-induced heart failure, mediated in part by a local impairment in mitochondrial function and increase in oxidative stress and myocyte apoptosis.     

Olmesartan, an AT1 antagonist, attenuates oxidative stress, endoplasmic reticulum stress and cardiac inflammatory mediators in rats with heart failure induced by experimental autoimmune myocarditis

Studies have demonstrated that angiotensin II has been involved in immune and inflammatory responses which might contribute to the pathogenesis of immune-mediated diseases. Recent evidence suggests that oxidative stress may play a role in myocarditis. Here, we investigated whether olmesartan, an AT(1)R antagonist protects against experimental autoimmune myocarditis (EAM) by suppression of oxidative stress, endoplasmic reticulum (ER) stress and inflammatory cytokines. EAM was induced in Lewis rats by immunization with porcine cardiac myosin, were divided into two groups and treated with either olmesartan (10 mg/kg/day) or vehicle for a period of 21 days. Myocardial functional parameters measured by hemodynamic and echocardiographic analyses were significantly improved by the treatment with olmesartan compared with those of vehicle-treated rats. Treatment with olmesartan attenuated the myocardial mRNA expressions of proinflammatory cytokines, [Interleukin (IL)-1β, monocyte chemoattractant protein-1, tumor necrosis factor-α and interferon-γ)] and the protein expression of tumor necrosis factor-α compared with that of vehicle-treated rats. Myocardial protein expressions of AT(1)R, NADPH oxidase subunits (p47phox, p67phox, gp91phox) and the expression of markers of oxidative stress (3-nitrotyrosine and 4-hydroxy-2-nonenal), and the cardiac apoptosis were also significantly decreased by the treatment with olmesartan compared with those of vehicle-treated rats. Furthermore, olmesartan treatment down-regulated the myocardial expressions of glucose regulated protein-78, growth arrest and DNA damage-inducible gene, caspase-12, phospho-p38 mitogen-activated protein kinase (MAPK) and phospho-JNK. These findings suggest that olmesartan protects against EAM in rats, at least in part via suppression of oxidative stress, ER stress and inflammatory cytokines.     

Visfatin aggravates transverse aortic constriction-induced cardiac remodelling by enhancing macrophage-mediated oxidative stress in mice

Previous studies have reported that visfatin can regulate macrophage polarisation, which has been demonstrated to participate in cardiac remodelling. The aims of this study were to investigate whether visfatin participates in transverse aortic constriction (TAC)-induced cardiac remodelling by regulating macrophage polarisation. First, TAC surgery and angiotensin II (Ang II) infusion were used to establish a mouse cardiac remodelling model, visfatin expression was measured, and the results showed that TAC surgery or Ang II infusion increased visfatin expression in the serum and heart in mice, and phenylephrine or hydrogen peroxide promoted the release of visfatin from macrophages in vitro. All these effects were dose-dependently reduced by superoxide dismutase. Second, visfatin was administered to TAC mice to observe the effects of visfatin on cardiac remodelling. We found that visfatin increased the cross-sectional area of cardiomyocytes, aggravated cardiac fibrosis, exacerbated cardiac dysfunction, further regulated macrophage polarisation and aggravated oxidative stress in TAC mice. Finally, macrophages were depleted in TAC mice to investigate whether macrophages mediate the regulatory effect of visfatin on cardiac remodelling, and the results showed that the aggravating effects of visfatin on oxidative stress and cardiac remodelling were abrogated. Our study suggests that visfatin enhances cardiac remodelling by promoting macrophage polarisation and enhancing oxidative stress. Visfatin may be a potential target for the prevention and treatment of clinical cardiac remodelling.     

Protective effect of alpha-tocopherol on oxidative stress in experimental pulmonary fibrosis in rats

The study was undertaken to investigate the influence of alpha-tocopherol (vitamin E) on malondialdehyde (MDA) and glutathione (GSH) levels and catalase (CAT) activity in lung of rats with bleomycin-induced pulmonary fibrosis (PF). Fourteen Wistar-albino rats were randomly divided into two groups of seven animals each. The first group was treated intra-tracheally with bleomycin hydrochloride (BM group); the second group was also instilled with BM but received injections of alpha-tocopherol twice a week (BM + E group). The third group was treated in the same manner with saline solution only, acting as controls (C). There were decreases in GSH level and CAT activity while an increase in MDA level in BM group was found compared to the control group (p < 0.05). Vitamin E had a regulator effect on these parameters. After administration of alpha-tocopherol, the increase in GSH level and CAT activity and the decrease in MDA level were seen in BM + E group compared to BM group (p < 0.05). Distinct histopathological changes were found in the BM group compared to the untreated rats. Less severe fibrotic lesions were also observed in the BM + E group. The results show that vitamin E is effective on the prevention of BM-induced PF, as indicated by differences in the lung levels of oxidants and antioxidants.     

益气化湿通络方对实验性肾功能衰竭大鼠肾脏氧化应激损伤及纤维化的改善作用

目的:观察益气化湿通络方对5/6肾切除肾衰竭模型大鼠残留肾脏氧化应激损伤及纤维化的改善作用。方法:采用Platt法建立5/6肾切除慢性肾衰竭大鼠模型。术后2周抽检大鼠确认造模成功后,将大鼠随机分为:模型组(Model)、益气化湿通络方组(YHT)、贝那普利组(BH)、假手术组(Sham),每组8只。每日灌胃治疗1次(YHT组免煎颗粒水溶液0.276 g/100 g;BH组盐酸贝那普利片剂水溶液0.09 mg/100 g灌胃;Sham及Model 1 ml/100 g生理盐水灌胃),连续治疗12周。12周末用代谢笼收集24 h尿液,检测尿蛋白含量。之后麻醉大鼠腹主动脉取血、摘取肾脏,检测血清血肌酐(Scr)、血尿素氮(BUN)含量;HE、Masson染色观察左肾病理改变;检测肾组织匀浆超氧化物歧化酶(SOD)的活性和丙二醛(MDA)的含量,检测肾组织中核因子NF-E2相关因子(Nrf2)、Kelch样环氧氯丙烷相关蛋白-1(Keap1)、NADPH氧化酶4(Nox4)、转化生长因子-β1(TGF-β1)、I型胶原蛋白(Collagen1)的表达以及Nrf2在肾组织细胞核内的表达。结果:与Sham组比较,Model组大鼠肾小球损伤较重,纤维化明显;Scr、BUN、MDA水平和24 h尿蛋白的排出量,Keap1、Nox4、TGF-β1、Collagen1的蛋白表达均明显升高(P＜0.01),SOD活性、Nrf2表达明显降低(P＜0.01);与Model组比较,经YHT或BH干预后肾小球病变程度减轻,纤维化较少,Scr、BUN、MDA水平和24 h尿蛋白的排出量,Keap1、Nox4、TGF-β1、Collagen1的蛋白表达均明显减少(P＜0.01),SOD活性、Nrf2表达明显升高(P＜0.01)。结论:益气化湿通络方通过影响Nrf2/Keap1信号通路、下调TGF-β1蛋白表达,从而改善肾衰竭模型大鼠残留肾脏的氧化应激损伤及纤维化程度。     

Effects of aerobic and resistance exercise on cardiac remodelling and skeletal muscle oxidative stress of infarcted rats

We compared the influence of aerobic and resistance exercise on cardiac remodelling, physical capacity and skeletal muscle oxidative stress in rats with MI‐induced heart failure. Three months after MI induction, Wistar rats were divided into four groups: Sham; sedentary MI (S‐MI); aerobic exercised MI (A‐MI); and resistance exercised MI (R‐MI). Exercised rats trained three times a week for 12 weeks on a treadmill or ladder. Statistical analysis was performed by ANOVA or Kruskal‐Wallis test. Functional aerobic capacity was greater in A‐MI and strength gain higher in R‐MI. Echocardiographic parameters did not differ between infarct groups. Reactive oxygen species production, evaluated by fluorescence, was higher in S‐MI than Sham, and lipid hydroperoxide concentration was lower in A‐MI than the other groups. Glutathione peroxidase activity was higher in A‐MI than S‐MI and R‐MI. Superoxide dismutase was lower in S‐MI than Sham and R‐MI. Gastrocnemius cross‐sectional area, satellite cell activation and expression of the ubiquitin‐proteasome system proteins did not differ between groups. In conclusion, aerobic exercise and resistance exercise improve functional capacity and maximum load carrying, respectively, without changing cardiac remodelling in infarcted rats. In the gastrocnemius, infarction increases oxidative stress and changes antioxidant enzyme activities. Aerobic exercise reduces oxidative stress and attenuates superoxide dismutase and glutathione peroxidase changes.     

Indoxyl sulfate promotes cardiac fibrosis with enhanced oxidative stress in hypertensive rats

AimsCardiovascular disease (CVD) is common in chronic kidney disease (CKD) patients. Indoxyl sulfate (IS) is a nephrovascular uremic toxin that induces oxidative stress in kidney and vascular system. The present study aimed to examine the effect of IS on fibrosis and oxidative stress in rat heart.Main methodsThe effects of IS on heart were examined by Masson's trichrome (MT) staining and immunohistochemistry using: (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive IS-administered rats (DN + IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive IS-administered rats (DH + IS).Key findingsDH + IS rats showed significantly increased heart weight and left ventricle weight compared with DN. DH and DH + IS rats showed significantly increased diameter of cardiomyocytes, increased MT-positive fibrotic area, increased staining for transforming growth factor (TGF)-β1, α-smooth muscle actin (SMA), type 1 collagen, NADPH oxidase Nox 4, malondialdehyde (MDA), and 8-hydroxydeoxyguanosine (8-OHdG) and decreased staining for nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1) in the heart compared with DN. More notably, DH + IS rats showed significantly increased diameter of cardiomyocytes, increased fibrotic area, increased staining for TGF-β1, SMA, type 1 collagen, Nox4, 8-OHdG and MDA, and decreased staining for Nrf2 and HO-1 in the heart compared with DH.SignificanceIS aggravates cardiac fibrosis and cardiomyocyte hypertrophy with enhanced oxidative stress and reduced anti-oxidative defense in hypertensive rats.     

Loss of cardiac tetralinoleoyl cardiolipin in human and experimental heart failure

The mitochondrial phospholipid cardiolipin is required for optimal mitochondrial respiration. In this study, cardiolipin molecular species and cytochrome oxidase (COx) activity were studied in interfibrillar (IF) and subsarcolemmal (SSL) cardiac mitochondria from Spontaneously Hypertensive Heart Failure (SHHF) and Sprague-Dawley (SD) rats throughout their natural life span. Fisher Brown Norway (FBN) and young aortic-constricted SHHF rats were also studied to investigate cardiolipin alterations in aging versus pathology. Additionally, cardiolipin was analyzed in human hearts explanted from patients with dilated cardiomyopathy. A loss of tetralinoleoyl cardiolipin (L(4)CL), the predominant species in the healthy mammalian heart, occurred during the natural or accelerated development of heart failure in SHHF rats and humans. L(4)CL decreases correlated with reduced COx activity (no decrease in protein levels) in SHHF cardiac mitochondria, but with no change in citrate synthase (a matrix enzyme) activity. The fraction of cardiac cardiolipin containing L(4)CL became much lower with age in SHHF than in SD or FBN mitochondria. In summary, a progressive loss of cardiac L(4)CL, possibly attributable to decreased remodeling, occurs in response to chronic cardiac overload, but not aging alone, in both IF and SSL mitochondria. This may contribute to mitochondrial respiratory dysfunction during the pathogenesis of heart failure.     

Induction of oxidative stress and disintegrin metalloproteinase in human heart end-stage failure

Collagen degradation is required for the creation of new integrin binding sites necessary for cell survival. However, a complete separation between the matrix and the cell leads to apoptosis, dilatation, and failure. Previous studies have demonstrated increased metalloproteinase activity in the failing myocardium. To test the hypothesis that disintegrin metalloproteinase (DMP) is induced in human heart end-stage failure, left ventricle tissue from ischemic cardiomyopathic (ICM, n = 10) and dilated cardiomyopathic (DCM, n = 10) human hearts were obtained at the time of orthotopic cardiac transplant. Normal (n = 5) tissue specimens were obtained from unused hearts. The levels of reduced oxygen species (ROS) were 12 +/- 2, 25 +/- 3, and 16 +/- 2 nmol (means +/- SE, P < 0.005) in normal, ICM, and DCM, respectively, by spectrofluorometry. The percent levels of endothelial cells were 100 +/- 15, 35 +/- 19, and 55 +/- 11 in normal, ICM, and DCM, respectively, by CD31 labeling. The levels of nitrotyrosine by Western analysis were significantly increased, and endothelial nitric oxide (NO) by the Griess method was decreased in ICM and DCM compared with normal tissue. The synthesis and degradation of beta(1)-integrin and connexin 43 were significantly increased in ICM and DCM compared with normal hearts by Western analysis. Levels of DMP were increased, and levels of cardiac inhibitor of metalloproteinase (CIMP) were decreased. Aggrecanase activity of DMP was significantly increased in ICM and DCM hearts compared with normal. These results suggest that the occurrence of cardiomyopathy is significantly confounded by the increase in ROS, nitrotyrosine, and DMP activity. This increase is associated with decreased NO, endothelial cell density, and CIMP. In vitro, treatment of CIMP abrogated the DMP activity. The treatment with CIMP may prevent degradation of integrin and connexin and ameliorate heart failure.     

Inhibition of NADPH oxidase reduces myocardial oxidative stress and apoptosis and improves cardiac function in heart failure after myocardial infarction

Increases in NADPH oxidase activity, oxidative stress, and myocyte apoptosis coexist in failing hearts. In cardiac myocytes in vitro inhibition of NADPH oxidase reduces apoptosis. In this study, we tested the hypothesis that NADPH oxidase inhibition reduces myocyte apoptosis and improves cardiac function in heart failure after myocardial infarction (MI). Rabbits with heart failure induced by MI and sham-operated animals were randomized to orally receive apocynin, an inhibitor of NADPH oxidase (15 mg per day) or placebo for 4 weeks. Left ventricular (LV) dimension and function were assessed by echocardiography and hemodynamics. Myocardial NADPH oxidase activity was measured by superoxide dismutase-inhibitable cytochrome c reduction assay, NADPH oxidase subunit p47phox expression by Western blot and immunofluorescence analysis, myocardial oxidative stress evaluated by 8-hydroxydeoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal (4-HNE) using immunohistochemistry, and myocyte apoptosis by TUNEL assay. MI rabbits exhibited LV dilatation and systolic dysfunction measured by LV fractional shortening and the maximal rate of LV pressure rise (dP/dt). These changes were associated with increases in NADPH oxidase activity, p47phox protein expression, 8-OHdG expression, 4-HNE expression, myocyte apoptosis, and Bax protein and a decrease in Bcl-2 protein. Apocynin reduced NADPH oxidase activity, p47phox protein, oxidative stress, myocyte apoptosis, and Bax protein, increased Bcl-2 protein, and ameliorated LV dilatation and dysfunction after MI. The results suggest that inhibition of NADPH oxidase may represent an attractive therapeutic approach to treat heart failure.     

Early activation of cardiac and renal endothelin systems in experimental heart failure

We investigated the time course of the expression of cardiac and renal endothelin systems in tachycardia-induced heart failure in dogs. Eleven beagles underwent rapid pacing at a progressively increased rate over a period of 5 wk, with a weekly clinical examination, echocardiography, measurement of circulating and urinary endothelin-1 (ET-1), and myocardial and renal tissue biopsies. Real-time quantitative PCR was used for determinations of tissue prepro-ET-1 (ppET-1), ET-1-converting enzyme (ECE-1), and ETA and ETB receptor mRNA. Cardiac and renal tissue ET-1 contents were evaluated by immunostaining and measured by radioimmunoassay at autopsy. Rapid pacing caused a progressive increase in end-systolic and end-diastolic ventricular volumes (P < 0.05) from week 2 together with a decrease in ejection fraction and in mean velocity of circumferential shortening (P < 0.05) from week 1. These changes were tightly correlated to myocardial ppET-1 and renal ETA receptor mRNA and less so to myocardial ECE-1 mRNA, and they occurred before any increase in plasma and urinary ET-1 (P < 0.05 from week 4) and clinical signs of heart failure. Renal ppET-1 did not change. Both cardiac and renal ET-1 peptide contents were increased at autopsy. We conclude that tachycardia-induced heart failure in dogs is characterized by an early activation of the cardiac and renal tissue endothelin systems, which occurs before any changes in circulating and urinary ET-1 and is closely related to altered ventricular function.     

Regulation of cardiac adrenomedullin-mRNA in different stages of experimental heart failure

Adrenomedullin (AM) is a peptide hormone with vasodilating and natriuretic properties. AM plasma concentrations are elevated in heart failure. Whether cardiac AM-mRNA synthesis is increased in heart failure is not known. We measured AM-mRNA/GAPDH-mRNA in all four heart chambers in compensated and overt heart failure in rats with two different sizes of aortocaval shunt. Left and right atrial AM-mRNA expressions were unchanged in both heart failure models. Similarly, left and right ventricular AM-mRNA expressions were unchanged in compensated heart failure. In overt heart failure, however, the AM-mRNA expression was significantly increased in the left ventricle (145+/-20 vs. 100+/-3% of control, p<0.05). The right ventricular AM-mRNA expression was significantly increased only in a subgroup of animals with pulmonary congestion (lung weight >2.0 g, 141+/-16 vs. 100+/-11% of control, p<0.05). Ventricular AM concentrations were elevated in both ventricles in overt heart failure. AM plasma concentrations were significantly higher in the subgroup with pulmonary congestion than in rats with compensated heart failure (496+/-95 vs. 143+/-7 pmol/l, p<0.01). These data indicate that ventricular AM-mRNA expression and AM concentrations were upregulated only in advanced stages of heart failure. However, the exact contribution of cardiac AM synthesis to the increased AM plasma levels remains to be established.     

Protective effects of resveratrol on calcium-induced oxidative stress in rat heart mitochondria

Trans-resveratrol is a nutraceutical with known antioxidant, anti-inflammatory, cardioprotective, and anti-apoptotic properties. The aim of this study was to evaluate the effects of resveratrol on heart mitochondria. Resveratrol significantly decreased Fe²⁺ + ascorbate oxidant system-induced lipid peroxide levels, preserved physiological levels of glutathione, and increased nitric oxide (NO) levels in mitochondria. Under calcium-mediated stress, there was a 2.7-fold increase in the NO levels, and a mild decoupling in the mitochondrial respiratory chain. These results provide a mechanism for and support the beneficial effects of resveratrol under pathological conditions induced by oxidative stress and calcium overload. In addition, these findings underscore the usefulness of resveratrol in the prevention of cardiovascular diseases.     

Uncompensated mitochondrial oxidative stress underlies heart failure in an iPSC-derived model of congenital heart disease

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Short-term exercise worsens cardiac oxidative stress and fibrosis in 8-month-old db/db mice by depleting cardiac glutathione

Abstract

Moderate exercise improves cardiac antioxidant status in young humans and animals with Type-2 diabetes (T2D). Given that both diabetes and advancing age synergistically decrease antioxidant expression in most tissues, it is unclear whether exercise can upregulate cardiac antioxidants in chronic animal models of T2D. To this end, 8-month-old T2D and normoglycemic mice were exercised for 3 weeks, and cardiac redox status was evaluated. As expected, moderate exercise increased cardiac antioxidants and attenuated oxidative damage in normoglycemic mice. In contrast, similar exercise protocol in 8-month-old db/db mice worsened cardiac oxidative damage, which was associated with a specific dysregulation of glutathione (GSH) homeostasis. Expression of enzymes for GSH biosynthesis [γ-glutamylcysteine synthase, glutathione reductase] as well as for GSH-mediated detoxification (glutathione peroxidase, glutathione-S-transferase) was lower, while toxic metabolites dependent on GSH for clearance (4-hydroxynonenal) were increased in exercised diabetic mice hearts. To validate GSH loss as an important factor for such aggravated damage, daily administration of GSH restored cardiac GSH levels in exercised diabetic mice. Such supplementation attenuated both oxidative damage and fibrotic changes in the myocardium. Expression of transforming growth factor beta (TGF-β) and its regulated genes which are responsible for such profibrotic changes were also attenuated with GSH supplementation. These novel findings in a long-term T2D animal model demonstrate that short-term exercise by itself can deplete cardiac GSH and aggravate cardiac oxidative stress. As GSH administration conferred protection in 8-month-old diabetic mice undergoing exercise, supplementation with GSH-enhancing agents may be beneficial in elderly diabetic patients undergoing exercise.     

Curcumin protects cardiac cells against ischemia-reperfusion injury: effects on oxidative stress, NF-kappaB, and JNK pathways

In this study we explored the effects of curcumin in cardiac cells subjected to a protocol simulating ischemia-reperfusion (IR). Curcumin (10 microM) was administered before ischemia (pretreatment) or at the moment of reperfusion (posttreatment) and its effects were compared to those produced by a reference antioxidant (Trolox) with an equal antioxidant capacity. IR cardiac cells showed clear signs of oxidative stress, impaired mitochondrial activity, and a marked development of both necrotic and apoptotic processes; at the same time, increases in NF-kappaB nuclear translocation and JNK phosphorylation were observed. Curcumin pretreatment was revealed to be the most effective in attenuating all these modifications and, in particular, in reducing the death of IR cells. This confirms that the protective effect of curcumin is not related simply to its antioxidant properties but involves other mechanisms, notably interactions in the NF-kappaB and JNK pathways. These findings suggest that curcumin administration, in particular before the hypoxic challenge, represents a promising approach to protecting cardiac cells against IR injury. In this scenario our results point out the importance of the chronology for the outcome of the treatment and provide a differential valuation of the degree of protection that curcumin can exert by its antioxidant activity or by other mechanisms.