Chemical evaluation of compounds as nitric oxide or peroxynitrite donors using the reactions with serotonin

It has been suggested that diabetes induces an increase in oxidative stress; the increased expression of heme-oxygenase 1 (HO-1) in liver is believed to be a sensitive marker of the stress response. The aim of this study was to examine whether diabetes is able to induce HO-1 expression in liver. The specific mRNA was amplified by RT/PCR and calibrated with amplified β-actin mRNA.

The mRNA HO-1 levels in the liver of spontaneously diabetic rats were increased by 1.8 fold compared with non diabetics; this supports the hypothesis of weak but significant oxidative damage due to chronic hyperglycaemia. This work represents the first in vivo study exploring the semi-quantitative expression of HO-1 in the liver of spontaneously diabetic rats.     

Adiponectin ameliorates hyperglycemia-induced cardiac hypertrophy and dysfunction by concomitantly activating Nrf2 and Brg1

Hyperglycemia-induced oxidative stress is implicated in the development of cardiomyopathy in diabetes that is associated with reduced adiponectin (APN) and heme oxygenase-1 (HO-1). Brahma-related gene 1 (Brg1) assists nuclear factor-erythroid-2-related factor-2 (Nrf2) to activate HO-1 to increase myocardial antioxidant capacity in response to oxidative stress. We hypothesized that reduced adiponectin (APN) impairs HO-1 induction which contributes to the development of diabetic cardiomyopathy, and that supplementation of APN may ameliorate diabetic cardiomyopathy by activating HO-1 through Nrf2 and Brg1 in diabetes. Control (C) and streptozotocin-induced diabetic (D) rats were untreated or treated with APN adenovirus (1×10⁹ pfu) 3 weeks after diabetes induction and examined and terminated 1 week afterward. Rat left ventricular functions were assessed by a pressure–volume conductance system, before the rat hearts were removed to perform histological and biochemical assays. Four weeks after diabetes induction, D rats developed cardiac hypertrophy evidenced as increased ratio of heart weight to body weight, elevated myocardial collagen I content, and larger cardiomyocyte cross-sectional area (all P<0.05 vs C). Diabetes elevated cardiac oxidative stress (increased 15-F_2t-isoprostane, 4-hydroxynonenal generation, 8-hydroxy-2′-deoxyguanosine, and superoxide anion generation), increased myocardial apoptosis, and impaired cardiac function (all P<0.05 vs C). In D rats, myocardial HO-1 mRNA and protein expression were reduced which was associated with reduced Brg1 and nuclear Nrf2 protein expression. All these changes were either attenuated or prevented by APN. In primarily cultured cardiomyocytes (CMs) isolated from D rats or in the embryonic rat cardiomyocytes cell line H9C2 cells incubated with high glucose (HG, 25 mM), supplementation of recombined globular APN (gAd, 2 μg/mL) reversed HG-induced reductions of HO-1, Brg1, and nuclear Nrf2 protein expression and attenuated cellular oxidative stress, myocyte size, and apoptotic cells. Inhibition of HO-1 by ZnPP (10 μM) or small interfering RNA (siRNA) canceled all the above gAd beneficial effects. Moreover, inhibition of Nrf2 (either by the Nrf2 inhibitor luteolin or siRNA) or Brg1 (by siRNA) canceled gAd-induced HO-1 induction and cellular protection in CMs and in H9C2 cells incubated with HG. In summary, our present study demonstrated that APN reduced cardiac oxidative stress, ameliorated cardiomyocyte hypertrophy, and prevented left ventricular dysfunction in diabetes by concomitantly activating Nrf2 and Brg1 to facilitate HO-1 induction.     

Suppression of NF-κB and NF-κB regulated oxidative stress and neuroinflammation by BAY 11-7082 (IκB phosphorylation inhibitor) in experimental diabetic neuropathy

Inflammation is an emerging patho-mechanism of diabetes and its complications. NF-κB pathway is one of the central machinery initiating and propagating inflammatory responses. The present study envisaged the involvement of NF-κB inflammatory cascade in the pathophysiology of diabetic neuropathy using BAY 11-7082, an IκB phosphorylation inhibitor. Streptozotocin was used to induce diabetes in Sprauge Dawley rats. BAY 11-7082 (1 &; 3 mg/kg) was administered to diabetic rats for 14 days starting from the end of six weeks post diabetic induction. Diabetic rats developed deficits in nerve functions and altered nociceptive parameters and also showed elevated expression of NF-κB (p65), IκB and p-IκB along with increased levels of IL-6 &; TNF-α and inducible enzymes (COX-2 and iNOS). Furthermore, there was an increase in oxidative stress and decrease in Nrf2/HO-1 expression. We observed that BAY 11-7082 alleviated abnormal sensory responses and deficits in nerve functions. BAY 11-7082 also ameliorated the increase in expression of NF-κB, IκB and p-IκB. BAY 11-7082 curbed down the levels of IL-6, TNF-α, COX-2 and iNOS in the sciatic nerve. Lowering of lipid peroxidation and improvement in GSH levels was also seen along with increased expression of Nrf2/HO-1. Thus it can be concluded that NF-κB expression and downstream expression of proinflammatory mediators are prominent features of nerve damage leading to inflammation and oxidative stress and BAY 11-7082 was able to ameliorate experimental diabetic neuropathy by modulating neuroinflammation and improving antioxidant defence.     

Induction of hemeoxygenase-1 reduces glomerular injury and apoptosis in diabetic spontaneously hypertensive rats

Induction of hemeoxygenase-1 (HO-1) lowers blood pressure and reduces organ damage in hypertensive animal models; however, a potential protective role for HO-1 induction against diabetic-induced glomerular injury remains unclear. We hypothesize that HO-1 induction will protect against diabetes-induced glomerular injury by maintaining glomerular integrity and inhibiting renal apoptosis, inflammation, and oxidative stress. Diabetes was induced with streptozotocin in spontaneously hypertensive rats (SHR) as a model where the coexistence of hypertension and diabetes aggravates the progression of diabetic renal injury. Control and diabetic SHR were randomized to receive vehicle or the HO-1 inducer cobalt protoporphyrin (CoPP). Glomerular albumin permeability was significantly greater in diabetic SHR compared with control, consistent with an increase in apoptosis and decreased glomerular nephrin and α(3)β(1)-integrin protein expression in diabetic SHR. CoPP significantly reduced albumin permeability and apoptosis and restored nephrin and α(3)β(1)-integrin protein expression levels in diabetic SHR. Glomerular injury in diabetic SHR was also associated with increases in NF-κB-induced inflammation and oxidative stress relative to vehicle-treated SHR, and CoPP significantly blunted diabetes-induced increases in glomerular inflammation and oxidative stress in diabetic SHR. These effects were specific to exogenous stimulation of HO-1, since incubation with the HO inhibitor stannous mesoporphyrin alone did not alter glomerular inflammatory markers or oxidative stress yet was able to prevent CoPP-mediated decreases in these parameters. These data suggest that induction of HO-1 reduces diabetic induced-glomerular injury and apoptosis and these effects are associated with decreased NF-κB-induced inflammation and oxidative stress.     

虫草发酵菌丝体对胰岛素抵抗大鼠氧化应激的缓解作用

探究虫草发酵菌丝体对胰岛素抵抗(IR)大鼠氧化应激的影响。采用低中高剂量虫草发酵菌丝体(剂量分别为1.65g/kg饲料,3.30g/kg饲料和6.60g/kg饲料)分别干预IR大鼠。口服葡萄糖耐量实验观察不同剂量虫草发酵菌丝体的降糖效果,同时观察血脂、氧化应激水平的变化情况。RT-PCR法测肌肉、肝脏组织氧化应激相关基因Nrf2、HO-1和NQO1mRNA相对表达量,Westernblot法测其蛋白相对表达量。结果发现CM干预能够显著改善IR大鼠空腹血糖、血脂和机体氧化应激水平,肌肉、肝脏组织Nrf2、HO-1和NQO1mRNA相对表达量显著上升,肝脏Nrf2和NQO1蛋白相对表达量显著上调,并且具有剂量依赖性。说明CM具有缓解IR大鼠氧化应激的作用。     

Heme oxygenase-1 prevents superoxide anion-associated endothelial cell sloughing in diabetic rats

Heme oxygenase-1 (HO-1) represents a key defense mechanism against oxidative injury. Hyperglycemia has been linked to increased oxidative stress, leading to endothelial dysfunction, delayed cell replication, and enhanced apoptosis. The effect of streptozotocin (STZ)-induced diabetes on HO activity, HO-1 promoter activity, superoxide anion (O*-2, and the number of circulating endothelial cells was measured. The expression of HO-1/HO-2 protein was unchanged, but HO activity was decreased in aortas of diabetic rats compared with control (p < 0.05). High glucose decreased HO-1 promoter activity (p < 0.05). Hyperglycemia increased O*-2 and this increase was augmented with HO-1 inhibition and diminished with HO-1 upregulation (p < 0.05). Circulating endothelial cells were significantly higher in diabetic rats and were decreased or increased with administration of the HO-1 inducer (CoPP) or inhibitor (SnMP), respectively (p<0.05). In conclusion, HO-1 upregulation in diabetic rats brings about an increase in serum bilirubin, a reduction in O*-2 production, and a decrease in endothelial cell sloughing.     

Supplementation with N-acetylcysteine and taurine failed to restore glutathione content in liver of streptozotocin-induced diabetics rats but protected from oxidative stress

Rats were rendered diabetic with streptozotocin and supplemented or not with N-acetylcysteine (NAC) and taurine (TAU). The liver was examined for the quantity of glutathione (GSH), both total and oxidised (GSSG), by HPLC assay. Moreover, the liver expression of gamma-glutamyl-cysteine synthetase, cysteine dioxygenase and heme oxygenase 1 was evaluated. Streptozotocin-diabetic rats showed decreased levels of liver glutathione (GSH); dietary supplementation with the antioxidants NAC and TAU failed to restore liver GSH to the level of control rats. Gamma-glutamyl-cysteine synthetase expression was not reduced in the diabetic rats, so the low hepatic GSH level in the supplemented diabetic rats cannot be ascribed to decreased expression of the biosynthetic key enzyme. Moreover, the diabetic rats showed no evidence of increased expression of cysteine dioxygenase, which could have indicated that NAC-derived cysteine was consumed in metabolic pathways different from GSH synthesis. However, NAC+TAU treatment provided partial protection from glutathione oxidation in the liver of diabetic rats; moreover, the antioxidant treatment reduced the hepatic overexpression of heme oxygenase 1 (HO-1) mRNA which was detected in the diabetic rats. In conclusion, although NAC was not able to restore liver GSH levels, the antioxidant treatment restrained GSH oxidation and HO-1 overexpression, which are markers of cellular oxidative stress: diabetic rats probably exploit NAC as an antioxidant itself rather than as a GSH precursor.     

Chronic tempol treatment attenuates the renal hemodynamic effects induced by a heme oxygenase inhibitor in streptozotocin diabetic rats

Heme oxygenase-1 (HO-1) is induced by oxidative stress and plays an important role in protecting the kidney from oxidant-mediated damage in the streptozotocin (STZ) rat model of type-1 diabetes mellitus (DM-1). HO-derived metabolites, presumably carbon monoxide (CO), mediate vasodilatory influences in the renal circulation, particularly in conditions linked to elevated HO-1 protein expression or diminished nitric oxide (NO) levels. We tested the hypothesis that diabetes increases oxidative stress and induces HO-1 protein expression, which contributes to regulate renal hemodynamics in conditions of low NO bioavailability. Two weeks after the induction of diabetes with STZ (65 mg/kg iv), Sprague-Dawley rats exhibited higher renal HO-1 protein expression, hyperglycemia, and elevated renal nitrotyrosine levels than control normoglycemic animals. In anesthetized diabetic rats, renal vascular resistance (RVR) was increased, and in vivo cortical NO levels were reduced (P < 0.05) compared with control animals. Acute administration of the HO inhibitor Stannous mesoporphyrin (SnMP; 40 μmol/kg iv) did not alter renal hemodynamics in control rats, but greatly decreased glomerular filtration rate and renal blood flow, markedly increasing RVR in hyperglycemic diabetic rats. Chronic oral treatment with the SOD mimetic tempol prevented the elevation of nitrotyrosine, the HO-1 protein induction, and the increases in RVR induced by SnMP in the diabetic group, without altering basal NO concentrations or RVR. Increasing concentrations of a CO donor (CO-releasing molecule-A1) on pressurized renal interlobar arteries elicited a comparable relaxation in vessels taken from control or diabetic animals. These results suggest that oxidative stress-induced HO-1 exerts vasodilatory actions that partially maintain renal hemodynamics in uncontrolled DM-1.     

Overexpression of human heme oxygenase-1 attenuates endothelial cell sloughing in experimental diabetes

Heme oxygenase (HO)-1 represents a key defense mechanism against oxidative injury. Hyperglycemia produces oxidative stress and various perturbations of cell physiology. The effect of streptozotocin (STZ)-induced diabetes on aortic HO activity, heme content, the number of circulating endothelial cells, and urinary 8-epi-isoprostane PGF2alpha (8-Epi) levels in control rats and rats overexpressing or underexpressing HO-1 was measured. HO activity was decreased in hyperglycemic rats. Hyperglycemia increased urinary 8-Epi, and this increase was augmented in rats underexpressing HO-1 and diminished in rats overexpressing HO-1. The number of detached endothelial cells and O2- formation increased in diabetic rats and in hyperglycemic animals underexpressing HO-1 and decreased in diabetic animals overexpressing HO-1 compared with controls. These data demonstrate that HO-1 gene transfer in hyperglycemic rats brings about a reduction in O2- production and a decrease in endothelial cell sloughing. Upregulation of HO-1 decreases oxidant production and endothelial cell damage and shedding and may attenuate vascular complications in diabetes.     

基于氧化应激研究苦豆子不同提取物的肝毒性机制

基于亚急性毒性实验对苦豆子不同提取物肝毒性机制进行研究,为苦豆子临床安全使用提供理论依据。分别采用75%乙醇回流法(ER)、水煎煮法(WD)、75%乙醇超声法(EU)和水超声法(WU)制备苦豆子提取物,通过不同提取物的亚急性毒性实验测定大鼠肝组织中谷丙转氨酶(ALT)、谷草转氨酶(AST)、超氧化物歧化酶(SOD)、谷胱甘肽酶(GSH)、丙二醛(MDA)水平以评价其肝毒性;应用免疫组织化学和蛋白免疫印迹方法测定氧化应激相关蛋白红系衍生的核因子2相关因子(Nrf2)、血红素氧合酶1(HO-1)、超氧化物歧化酶1(SOD1)、超氧化物歧化酶2(SOD2)表达,进一步探讨其肝毒性机制。结果显示,与空白组相比,AST在雄性大鼠各给药组血清中显著性升高,ALT在雌雄大鼠血清中均呈升高趋势,尤其在雄性WD、ER组、雌性WD、WU、EU组中有显著性差异。与空白组比较,雄性给药大鼠肝脏SOD和GSH在各组中均显著性降低,GSH在雌性大鼠肝组织中呈升高趋势,其中WD组有显著性差异,MDA在雌、雄给药大鼠肝脏中均显著升高。给药后,对Nrf2/HO-1氧化应激通路中相关蛋白检测后发现,Nrf2蛋白相较空白组在肝组织中表达均呈降低趋势,其中雄性WD、WU、EU组和雌性WD、EU、ER组最为显著,HO-1在雌雄各组肝组织表达中均发生显著性降低。SOD1在雌性各组中均有显著性降低,而在雄性中WD和ER组有显著性降低。本研究发现苦豆子不同提取物对大鼠肝脏都有一定的毒性,其毒性机制主要是通过调控Nrf2/HO-1通路中相关蛋白造成机体氧化应激而发生。     

Tissue specific expression and immunohistochemical localization of glutathione S-transferase in streptozotocin induced diabetic rats: modulation by Momordica charantia (karela) extract

In streptozotocin (STZ)-induced diabetes, destruction of pancreatic beta-cell causes an acute shortage of insulin. Increased oxidative stress is believed to be one of the main factors in the etiology and complications of diabetes. In this study we have reported hyperglycemia and glutathione-associated oxidative stress in rats one week after treatment with STZ. In our previous studies, we have reported oxidative stress-related changes in xenobiotic metabolism in tissues from STZ-induced chronic diabetic rats. Here, we demonstrate by immunohistochemistry, that glutathione S-transferase (GST) isoenzymes are differentially expressed in the liver, kidney and testis of diabetic rats. The distribution of GST isoenzymes was found to be tissue- and regio-specific. In addition, we have also shown that treatment with an extract of Momordica charantia (karela), an antidiabetic herb, modulates GST expression in diabetic rats and reverts them to the normal distribution as seen in the tissues of control rats. These results suggest that glutathione metabolism and GST distribution in the tissues of diabetic rats may play an important role in the etiology, pathology and prevention of diabetes.     

The BET/BRD inhibitor JQ1 attenuates diabetes-induced cognitive impairment in rats by targeting Nox4-Nrf2 redox imbalance

Diabetes-induced oxidative damage is believed to play an important role in the development of cognitive dysfunction. In this study, the involvement of the Nox4-Nrf2 redox imbalance was investigated. STZ-induced diabetic rats exhibited obvious oxidative stress and apoptosis in the hippocampus assessed by augmentation of lipid peroxidation, positive TUNEL staining, elevated ratio of Bax/Bcl-2 and increased caspase 3 activity. Furthermore, hyperglycemia markedly increased Nox4 activity and reduced the activation of Nrf2 by suppressing its up-stream regulatory Akt as well as down-stream target HO-1. Significant improvement of cognitive performance was observed after treatment with the BET/BRD inhibitor JQ1, accompanied by decreased oxidative stress, neuroinflammation and apoptosis in the hippocampus. JQ1 treatment also improved changes in the neuronal cell morphology as well as increased the expression of p-AKT, Nrf2 and HO-1. Our results provide evidence indicating that JQ1 treatment could modulate Nox4-Nrf2 redox imbalance in the hippocampus and may be a promising agent for diabetes-associated cognitive dysfunction.     

A High Expression of Heme Oxygenase-1 in the Liver of LEC Rats at the Stage of Hepatoma: The Possible Implication of Induction in Uninvolved Tissue

We have examined changes in the expression of heme oxygenase-1 (HO-1), an inducible isoform and HO-2, a constitutive isoform, in the liver of Long-Evans with a Cinnamon-like color (LEC) rat, a mutant strain which spontaneously develops acute hepatitis and hepatoma. HO-1 expression was highly enhanced in the LEC rat livers with jaundice, and then decreased slightly, but overall remained at a higher level than in the Long-Evans with Agouti color (LEA) control rats, as judged by Northern blotting analysis of the whole liver extract. The high expression of HO-1 in the LEC rat liver was, however, not due to the actual cancer lesion but, rather, due to the surrounding uninvolved tissues including hepatocytes. Immunohistochemical analysis also supported this conclusion. Among normal tissues, the expression of HO-1 but not HO-2 was high in only the spleen of both LEC and LEA rats.

The high expression observed in the stage of acute hepatitis and hepatoma stages in the LEC rat is probably due to the oxidative stress caused by the accumulation of free copper and free iron levels which has been reported earlier by our group (Suzuki et al., Carcinogenesis, 1993,14, 1881-1884 and Koizumi et al., Free Radical Research, in press) as well as by free heme levels. The inflammatory cytokines produced by the surrounding tissue at the hepatoma stage would also be expected to play a role in the induction mechanism. The physiological relevance of HO-1 induction might be an adaptive response to oxidative stress and vasodilatory effect of carbon monoxide on sinusoidal circulation.     

Caffeic acid phenethyl ester (CAPE) protects brain against oxidative stress and inflammation induced by diabetes in rats

Diabetic patients reveal significant disorders, such as nephropathy, cardiomyopathy, and neuropathy. As oxidative stress and inflammation seem to be implicated in the pathogenesis of diabetic brain, we aimed to investigate the effects of caffeic acid phenethyl ester (CAPE) on oxidative stress and inflammation in diabetic rat brain. Diabetes was induced by a single dose of streptozotocin (45 mg kg⁻¹, i.p.) injection into rats. Two days after streptozotocin treatment 10 μM kg⁻¹ day⁻¹ CAPE was administrated and continued for 60 days. Here, we demonstrate that CAPE significantly decreased the levels of nitric oxide and malondialdehyde induced by diabetes, and the activities of catalase, glutathione peroxidase, and xanthine oxidase in the brain. However, glutathione levels were increased by CAPE. The mRNA expressions of tumor necrosis factor (TNF)-α and interferon (IFN)-γ, and inducible nitric oxide synthase (iNOS) were remarkably enhanced in brain by diabetes. CAPE treatments significantly suppressed these inflammatory cytokines (about 70% for TNF-α, 26% for IFN-γ) and NOS (completely). Anti-inflammatory cytokine IL-10 mRNA expression was not affected by either diabetes or CAPE treatments. In conclusion, diabetes induces oxidative stress and inflammation in the brain, and these may be contributory mechanisms involved in this disorder. CAPE treatment may reverse the diabetic-induced oxidative stress in rat brains. Moreover, CAPE reduces the mRNA expressions of TNF-α and IFN-γ in diabetic brain; suggesting CAPE suppresses inflammation as well as oxidative stress occurred in the brain of diabetic patients.     

The Sodium-Glucose Co-Transporter 2 Inhibitor Empagliflozin Improves Diabetes-Induced Vascular Dysfunction in the Streptozotocin Diabetes Rat Model by Interfering with Oxidative Stress and Glucotoxicity

Objective

In diabetes, vascular dysfunction is characterized by impaired endothelial function due to increased oxidative stress. Empagliflozin, as a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i), offers a novel approach for the treatment of type 2 diabetes by enhancing urinary glucose excretion. The aim of the present study was to test whether treatment with empagliflozin improves endothelial dysfunction in type I diabetic rats via reduction of glucotoxicity and associated vascular oxidative stress.

Methods

Type I diabetes in Wistar rats was induced by an intravenous injection of streptozotocin (60 mg/kg). One week after injection empagliflozin (10 and 30 mg/kg/d) was administered via drinking water for 7 weeks. Vascular function was assessed by isometric tension recording, oxidative stress parameters by chemiluminescence and fluorescence techniques, protein expression by Western blot, mRNA expression by RT-PCR, and islet function by insulin ELISA in serum and immunohistochemical staining of pancreatic tissue. Advanced glycation end products (AGE) signaling was assessed by dot blot analysis and mRNA expression of the AGE-receptor (RAGE).

Results

Treatment with empagliflozin reduced blood glucose levels, normalized endothelial function (aortic rings) and reduced oxidative stress in aortic vessels (dihydroethidium staining) and in blood (phorbol ester/zymosan A-stimulated chemiluminescence) of diabetic rats. Additionally, the pro-inflammatory phenotype and glucotoxicity (AGE/RAGE signaling) in diabetic animals was reversed by SGLT2i therapy.

Conclusions

Empagliflozin improves hyperglycemia and prevents the development of endothelial dysfunction, reduces oxidative stress and improves the metabolic situation in type 1 diabetic rats. These preclinical observations illustrate the therapeutic potential of this new class of antidiabetic drugs.     

Therapeutic effect of green tea extract on oxidative stress in aorta and heart of streptozotocin diabetic rats

Hyperglycemia induced oxidative stress has been proposed as a cause of many complications of diabetes including cardiac dysfunction. The present study depicts the therapeutic effect of green tea extract on oxidative stress in aorta as well as heart of streptozotocin diabetic rats. Six weeks after diabetes induction, green tea was administered orally for 4 weeks [300 mg (kg body weight)(-1) day (-1)]. In aorta and heart of diabetic rats there was a significant increase in the activity of superoxide dismutase, catalase and glutathione peroxidase with an increase in lipid peroxides. Diabetic rats showed a significant decrease in the levels of serum and cardiac glutathione. Green tea administration to diabetic rats reduced lipid peroxides and activity of antioxidant enzymes whereas increased glutathione content. The results demonstrate that the induction of antioxidant enzymes in diabetic rats is not efficient and sufficient to reduce the oxidative stress. But green tea by providing a competent antioxidative mechanism ameliorates the oxidative stress in the aorta and heart of diabetic rats. The study suggests that green tea may provide a useful therapeutic option in the reversal of oxidative stress induced cardiac dysfunction in diabetes mellitus.