Antiarrhythmic effect of lithium in rats after myocardial infarction by activation of Nrf2/HO-1 signaling

Glycogen synthase kinase-3 (GSK-3) signaling has been shown to play a role in the regulation of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of antioxidant genes, including heme oxygenase-1 (HO-1). We assessed whether lithium, a GSK-3 inhibitor, attenuates cardiac sympathetic reinnervation after myocardial infarction, a status of high reactive oxygen species (ROS), by attenuating nerve growth factor (NGF) expression and whether Nrf2/HO-1 signaling is involved in the protection. Twenty-four hours after ligation of the left anterior descending artery, male Wistar rats were treated for 4 weeks. The postinfarction period was associated with increased oxidative–nitrosative stress, as measured by myocardial superoxide, nitrotyrosine, and dihydroethidium fluorescent staining. In concert, myocardial norepinephrine levels and immunohistochemical analysis of sympathetic nerve revealed a significant increase in innervation in vehicle-treated rats compared with sham-operated rats. Arrhythmic scores during programmed stimulation in the vehicle-treated rats were significantly higher than those in sham. This was paralleled by a significant upregulation of NGF protein and mRNA in the vehicle-treated rats, which was reduced after administration of LiCl. LiCl stimulated the nuclear translocation of Nrf2 and the transactivation of the Nrf2 target gene HO-1. Inhibition of phosphoinositide 3-kinase by wortmannin reduced the increase in Nrf2 nucleus translocation and HO-1 expression compared with lithium alone. In addition, the lithium-attenuated NGF levels were reversed in the presence of the Nrf2 inhibitor trigonelline, HO-1 inhibitor SnPP, and peroxynitrite generator SIN-1, indicating the role of Nrf2/HO-1/ROS. In conclusion, lithium protects against ventricular arrhythmias by attenuating NGF-induced sympathetic innervation via antioxidant activation of the Nrf2/HO-1 axis.     

Nrf2 prevents diabetic cardiomyopathy via antioxidant effect and normalization of glucose and lipid metabolism in the heart

Metabolic disorders and oxidative stress are the main causes of diabetic cardiomyopathy. Activation of nuclear factor erythroid 2-related factor 2 (Nrf2) exerts a powerful antioxidant effect and prevents the progression of diabetic cardiomyopathy. However, the mechanism of its cardiac protection and direct action on cardiomyocytes are not well understood. Here, we investigated in a cardiomyocyte-restricted Nrf2 transgenic mice (Nrf2-TG) the direct effect of Nrf2 on cardiomyocytes in DCM and its mechanism. In this study, cardiomyocyte-restricted Nrf2 transgenic mice (Nrf2-TG) were used to directly observe whether cardiomyocyte-specific overexpression of Nrf2 can prevent diabetic cardiomyopathy and correct glucose and lipid metabolism disorders in the heart. Compared to wild-type mice, Nrf2-TG mice showed resistance to diabetic cardiomyopathy in a streptozotocin-induced type 1 diabetes mouse model. This was primarily manifested as improved echocardiography results as well as reduced myocardial fibrosis, cardiac inflammation, and oxidative stress. These results showed that Nrf2 can directly act on cardiomyocytes to exert a cardioprotective role. Mechanistically, the cardioprotective effects of Nrf2 depend on its antioxidation activity, partially through improving glucose and lipid metabolism by directly targeting lipid metabolic pathway of AMPK/Sirt1/PGC-1α activation via upstream genes of sestrin2 and LKB1, and indirectly enabling AKT/GSK-3β/HK-Ⅱ activity via AMPK mediated p70S6K inhibition.     

Allopurinol reduces oxidative stress and activates Nrf2/p62 to attenuate diabetic cardiomyopathy in rats

Allopurinol (ALP) attenuates oxidative stress and diabetic cardiomyopathy (DCM), but the mechanism is unclear. Activation of nuclear factor erythroid 2‐related factor 2 (Nrf2) following the disassociation with its repressor Keap1 under oxidative stress can maintain inner redox homeostasis and attenuate DCM with concomitant attenuation of autophagy. We postulated that ALP treatment may activate Nrf2 to mitigate autophagy over‐activation and consequently attenuate DCM. Streptozotocin‐induced type 1 diabetic rats were untreated or treated with ALP (100 mg/kg/d) for 4 weeks and terminated after heart function measurements by echocardiography and pressure‐volume conductance system. Cardiomyocyte H9C2 cells infected with Nrf2 siRNA or not were incubated with high glucose (HG, 25 mmol/L) concomitantly with ALP treatment. Cell viability, lactate dehydrogenase, 15‐F2t‐Isoprostane and superoxide dismutase (SOD) were measured with colorimetric enzyme‐linked immunosorbent assays. ROS, apoptosis, was assessed by dihydroethidium staining and TUNEL, respectively. The Western blot and qRT‐PCR were used to assess protein and mRNA variations. Diabetic rats showed significant reductions in heart rate (HR), left ventricular eject fraction (LVEF), stroke work (SW) and cardiac output (CO), left ventricular end‐systolic volume (LVVs) as compared to non‐diabetic control and ALP improved or normalized HR, LVEF, SW, CO and LVVs in diabetic rats (all P < .05). Hearts of diabetic rats displayed excessive oxidative stress manifested as increased levels of 15‐F2t‐Isoprostane and superoxide anion production, increased apoptotic cell death and cardiomyocytes autophagy that were concomitant with reduced expressions of Nrf2, heme oxygenase‐1 (HO‐1) and Keap1. ALP reverted all the above‐mentioned diabetes‐induced biochemical changes except that it did not affect the levels of Keap1. In vitro, ALP increased Nrf2 and reduced the hyperglycaemia‐induced increases of H9C2 cardiomyocyte hypertrophy, oxidative stress, apoptosis and autophagy, and enhanced cellular viability. Nrf2 gene silence cancelled these protective effects of ALP in H9C2 cells. Activation of Nrf2 subsequent to the suppression of Keap1 and the mitigation of autophagy over‐activation may represent major mechanisms whereby ALP attenuates DCM.     

Diabetes aggravates myocardial ischaemia reperfusion injury via activating Nox2‐related programmed cell death in an AMPK‐dependent manner

Cardiovascular diseases such as myocardial ischaemia have a high fatality rate in patients with diabetes. This study was designed to expose the crosstalk between oxidative stress and AMPK, a vital molecule that controls biological energy metabolism, in myocardial ischaemia reperfusion injury (I/RI) in diabetic rats. Diabetes was stimulated in rats using streptozotocin injection. Rats were separated on random into control, control + I/R, Diabetes, Diabetes + I/R, Diabetes + I/R + N‐acetylcysteine and Diabetes + I/R + Vas2870 groups. Myocardial infarct size was determined, and the predominant Nox family isoforms were analysed. In vitro, the H9C2 cells were administered excess glucose and exposed to hypoxia/reoxygenation to mimic diabetes and I/R. The AMPK siRNA or AICAR was used to inhibit or activate AMPK expression in H9C2 cells, respectively. Then, myocardial oxidative stress and programmed cell death were measured. Diabetes or high glucose levels were found to aggravate myocardial I/RI or hypoxia/reoxygenation in H9C2 cells, as demonstrated by an increase in myocardial infarct size or lactate dehydrogenase levels, oxidative stress generation and induction of programmed cell death. In diabetic rat hearts, cardiac Nox1, Nox2 and Nox4 were all heightened. The suppression of Nox2 expression using Vas2870 or Nox2‐siRNA treatment in vivo or in vitro, respectively, protected diabetic rats from myocardial I/RI. AMPK gene knockout increased Nox2 protein expression while AMPK agonist decreased Nox2 expression. Therefore, diabetes aggravates myocardial I/RI by generating of Nox2‐associated oxidative stress in an AMPK‐dependent manner, which led to the induction of programmed cell death such as apoptosis, pyroptosis and ferroptosis.     

Modulation of Nrf2 expression alters high glucose-induced oxidative stress and antioxidant gene expression in mouse mesangial cells

Reactive oxygen species (ROS) play an important role in the pathogenesis of diabetic nephropathy. Nuclear factor erythroid 2-related factor 2 (Nrf2) can up-regulate the expression of antioxidant genes and protect cells from oxidative damage. The current study is aimed at examining the effect of modulation of Nrf2 expression on high glucose-induced oxidative stress and Nrf2-targeting antioxidant expression in mouse mesangial cells. In this study, mouse mesangial cells were transiently transfected with Nrf2-plasmid or the Nrf2-specific siRNA. The high glucose-induced intracellular ROS, malondialdehyde, cell proliferation, and TGF-β1 secretion were measured. The levels of Nrf2, heme oxygenase-1 (HO-1), γ-glutamylcysteine synthethase (γ-GCS) expression, and nuclear expression of Nrf2 in mouse mesangial cells were determined. We found that high glucose induced ROS and malondialdehyde generation in mouse mesangial cells. Induction of Nrf2 over-expression reduced the high glucose-induced ROS and malondialdehyde production, inhibited cell proliferation and TGF-β1 secretion, accompanied by up-regulating the expressions of HO-1 and γ-GCS in mouse mesangial cells. However, knockdown of Nrf2 expression displayed reverse effects in mouse mesangial cells. All these results indicated that Nrf2 and its downstream antioxidants, HO-1 and γ-GCS, are negative regulators of high glucose-induced ROS-related mouse mesangial cell dysfunction.     

Garlic Attenuates Cardiac Oxidative Stress via Activation of PI3K/AKT/Nrf2-Keap1 Pathway in Fructose-Fed Diabetic Rat

Background

Cardiovascular complication due to diabetes has remained a major cause of death. There is an urgent need to intervene the cardiac complications in diabetes by nutritional or pharmacological agents. Thus the present study was designed to find out the effectiveness of garlic on cardiac complications in insulin-resistant diabetic rats.

Methods and Results

SD rats were fed high fructose (65%) diet alone or along with raw garlic homogenate (250 mg/kg/day) or nutrient-matched (65% corn starch) control diet for 8 weeks. Fructose-fed diabetic rats showed cardiac hypertrophy, increased NFkB activity and increased oxidative stress. Administration of garlic significantly decreased (p<0.05) cardiac hypertrophy, NFkB activity and oxidative stress. Although we did not observe any changes in myocardial catalase, GSH and GPx in diabetic heart, garlic administration showed significant (p<0.05) increase in all three antioxidant/enzymes levels. Increased endogenous antioxidant enzymes and gene expression in garlic treated diabetic heart are associated with higher protein expression of Nrf2. Increased myocardial H₂S levels, activation of PI3K/Akt pathway and decreased Keap levels in fructose-fed heart after garlic administration might be responsible for higher Nrf2 levels.

Conclusion

Our study demonstrates that raw garlic homogenate is effective in reducing cardiac hypertrophy and fructose-induced myocardial oxidative stress through PI3K/AKT/Nrf2-Keap1 dependent pathway.     

Fisetin induces Nrf2-mediated HO-1 expression through PKC-δ and p38 in human umbilical vein endothelial cells

Fisetin is a natural flavonoid from fruits and vegetables that exhibits antioxidant, neurotrophic, anti-inflammatory, and anti-cancer effects in various disease models. Up-regulation of heme oxygenase-1 (HO-1) expression protects against oxidative stress-induced cell death, and therefore, plays a crucial role in cytoprotection in a variety of pathological models. In the present study, we investigated the effect of fisetin on the up-regulation of HO-1 in human umbilical vein endothelial cells (HUVECs). Small interfering RNA and pharmacological inhibitors of PKC-δ and p38 MAPK attenuated HO-1 induction in fisetin-stimulated HUVECs. Fisetin treatment resulted in significantly increased NF-E2-related factor 2 (Nrf2) nuclear translocation, and antioxidant response element (ARE)-luciferase activity, leading to up-regulation of HO-1 expression. In addition, fisetin pretreatment reduced hydrogen peroxide (H(2)O(2))-induced cell death, and this effect was reversed by ZnPP, an inhibitor of HO-1. In summary, these findings suggest that induction of HO-1 expression via Nrf2 activation may contribute to the cytoprotection exerted by fisetin against H(2)O(2) -induced oxidative stress in HUVECs.     

Induction of glutathione synthesis and heme oxygenase 1 by the flavonoids butein and phloretin is mediated through the ERK/Nrf2 pathway and protects against oxidative stress

Butein and phloretin are chalcones that are members of the flavonoid family of polyphenols. Flavonoids have well-known antioxidant and anti-inflammatory activities. In rat primary hepatocytes, we examined whether butein and phloretin affect tert-butylhydroperoxide (tBHP)-induced oxidative damage and the possible mechanism(s) involved. Treatment with butein and phloretin markedly attenuated tBHP-induced peroxide formation, and this amelioration was reversed by l-buthionine-S-sulfoximine [a glutamate cysteine ligase (GCL) inhibitor] and zinc protoporphyrin [a heme oxygenase 1 (HO-1) inhibitor]. Butein and phloretin induced both HO-1 and GCL protein and mRNA expression and increased intracellular glutathione (GSH) and total GSH content. Butein treatment activated the ERK1/2 signaling pathway and increased Nrf2 nuclear translocation, Nrf2 nuclear protein-DNA binding activity, and ARE-luciferase reporter activity. The roles of the ERK signaling pathway and Nrf2 in butein-induced HO-1 and GCL catalytic subunit (GCLC) expression were determined by using RNA interference directed against ERK2 and Nrf2. Both siERK2 and siNrf2 abolished butein-induced HO-1 and GCLC protein expression. These results suggest the involvement of ERK2 and Nrf2 in the induction of HO-1 and GCLC by butein. In an animal study, phloretin was shown to increase GSH content and HO-1 expression in rat liver and decrease carbon tetrachloride-induced hepatotoxicity. In conclusion, we demonstrate that butein and phloretin up-regulate HO-1 and GCL expression through the ERK2/Nrf2 pathway and protect hepatocytes against oxidative stress.     

基于PERK/Nrf2/HO-1信号通路研究瑞马唑仑对心肌缺血再灌注损伤大鼠铁死亡的影响

摘要 目的：基于蛋白激酶R样内质网激酶（PERK）/核因子E2相关因子2（Nrf2）/血红素氧合酶-1（HO-1）信号通路探究瑞马唑仑对心肌缺血再灌注损伤（MIRI）大鼠铁死亡的影响。方法：将90只SD大鼠随机分为假手术（Sham）组、MIRI组、低剂量-瑞马唑仑组（L-瑞马唑仑组,5 mg/kg）、高剂量-瑞马唑仑组（H-瑞马唑仑组,20 mg/kg）、H-瑞马唑仑+PERK抑制剂组（瑞马唑仑20 mg/kg+GSK2606414 1 mg/kg）,每组18只。采用结扎冠状动脉左前降支（LAD）0.5 h、再灌注2 h制备MIRI大鼠模型,于再灌注2 h后即刻尾静脉注射给药,再灌注24 h后进行组织取材。酶联免疫吸附（ELISA）法检测血清心肌损伤标志物[肌酸激酶同工酶（CK-MB）、心肌肌钙蛋白I（cTnI）]水平;HE染色观察心肌组织病理改变;Tunel染色检测心肌细胞凋亡;透射电镜观察心肌细胞超微结构变化;检测心肌组织中铁死亡相关标志物[铁、活性氧（ROS）、谷胱甘肽（GSH）、丙二醛（MDA）]水平;蛋白质印迹法（Western Blot）检测心肌组织中PERK/Nrf2/HO-1信号通路相关蛋白表达。结果：与Sham组相比,MIRI组心肌结构受损,纤维排列紊乱,线粒体呈现显著的铁死亡特征（膜固缩,膜密度增加,嵴减少）,血清中CK-MB、cTnI水平,心肌细胞凋亡率及心肌组织中铁、ROS、MDA水平升高（P<0.05）,心肌组织中GSH水平及p-PERK/PERK、核Nrf2/Nrf2、HO-1蛋白表达降低（P<0.05）;与MIRI组相比,L-瑞马唑仑组和H-瑞马唑仑组心肌组织上述病理改变明显减轻,血清CK-MB、cTnI水平,心肌细胞凋亡率及心肌组织中铁、ROS、MDA水平降低（P<0.05）,心肌组织中GSH水平及p-PERK/PERK、核Nrf2/Nrf2、HO-1蛋白表达升高（P<0.05）;与H-瑞马唑仑组相比,H-瑞马唑仑+PERK抑制剂组心肌组织上述病理改变加重,血清CK-MB、cTnI水平,心肌细胞凋亡率及心肌组织中铁、ROS、MDA水平升高（P<0.05）,心肌组织中GSH水平及p-PERK/PERK、核Nrf2/Nrf2、HO-1蛋白表达降低（P<0.05）。结论：瑞马唑仑可通过抑制铁死亡减轻大鼠MIRI,可能通过激活PERK/Nrf2/HO-1信号通路而实现。