Sprague-Dawley male rats aged 8 weeks and weighing 250 to 280 gram were purchased from the Animal Unit of Wuhan University, China, and allowed to adapt in their cages for three days before experiments. All rats had free access to standard chow and water. The experiments were performed in accordance with our institutional animal care guidelines which are in line with the use of Laboratory Animals published by the US National Institutes of Health. The study was approved by Institutional Animal Care and Use Committee.

Diabetes was induced by a single tail vein injection (under halothane anaesthesia) of streptozotocin (Sigma, St. Louis, MO, USA) (STZ, 60 mg/kg body weight, freshly dissolved in 0.1 M citrate buffer, pH 4.5), while control rats (n=7) were injected equal volume citrate buffer alone. Three days following STZ injection, blood glucose levels were measured using a Glucose Analyzer (Beckman Instruments, Fullerton, CA, USA), and rats with hyperglycemia (set at plasma glucose ≥16.7 mM) were used for the experiments.

NAC, at the dose of 1.5 g/kg/day, had no effects on NADPH oxidase activity and SOD activity in non-diabetic control rats but had effects on these parameters in diabetic rats.16 In the current study, therefore, NAC was used only in diabetic rats. One week after the induction of diabetes, rats were randomly assigned into NAC-treated or untreated groups (n=7, per group), and they were treated respectively with the antioxidant NAC (1.5 g/kg/day, D+NAC) or an equal volume of 0.9% saline (D) delivered by oral gavage. Rats in the non-diabetic control group (C) were treated also with an equal volume of 0.9% saline delivered by oral gavage. Treatments in all groups were continued for four weeks.

At the end of four weeks of treatment, the rats were anti-coagulated with heparin (1000 IU/kg) and then anaesthetized with pentobarbital sodium (65 mg/kg body weight). Blood glucose level and body weight were measured by a Glucose Analyzer (Beckman Instruments, Fullerton, CA, USA) and a lab scale, respectively. Blood samples were collected in heparinized syringes by heart puncture after opening the thoracic cavity. The collected blood was immediately centrifuged at 3000×g, plasma separated, aliquoted into small microcentrifuge tubes, and stored at -80℃ for further detection. Heart, kidney, liver and lung tissues were harvested immediately after sacrifice and frozen in liquid nitrogen and stored at -80℃ for further experiments.

Plasma levels of inflammatory cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 were measured, using the commercially available rat ELISA kit (Bender Med, Vienna, Austria) according to the manufacturer's instructions.

15-F_2t-isoprostane (15-F_2t-IsoP), a specific marker of oxidative stress, was measured using an EIA kit (Cayman chemical, Ann Arbor, MI, USA). Thus, plasma samples or homogenized heart tissue (in PBS) were purified using Affinity Sorbent/Column (Cayman chemical, Ann Arbor, MI, USA) in the presence of 0.01% butylated hydroxytoluene and then processed for analysis of free 15-F_2t-IsoP as described.26 The values of plasma or tissue 15-F_2t-IsoP were expressed as pg/mL in plasma or pg/mg protein in tissue homogenates.

Tissue sections (50 mg) were homogenized in 1 mL of cold buffer (5 mM potassium phosphate, pH 7.4, containing 0.9% sodium chloride and 0.1% glucose) and were centrifuged at 10000×g for 15 min at 4℃. The supernatant was collected for assay. Total antioxidant activity in plasma and tissue homogenate was measured using the Total Antioxidant Status Assay Kit (Calbiochem, Darmstadt, Germany), which is dependent on the capacity in the sample to inhibit the oxidation of ABTS™ [2,2'-Azino-di-(3-ethylbenz-thiazoline sulphonate)] to ABTS™^·+ by metmyoglobin (a peroxidase). The amount of ABTS™^·+ produced can be monitored by reading absorbance at 600 nm. Under the reaction conditions used, the antioxidants in the sample suppress absorbance at 600 nm by the degree proportional to their concentration. Total antioxidant activity was calculated and expressed as millimole per liter (mM) in plasma or millimole per gram protein in tissue samples (mmol/g protein).

SOD activity was determined using a Superoxide Dismutase Assay Kit (Cayman chemical, Ann Arbor, MI, USA) following the manufacturer's instructions. Plasma samples were assayed immediately after being thawed. Tissue sections (50 mg) were homogenized in 1 mL of cold 20 mM HEPES buffer, pH 7.2, containing 1 mM EGTA, 210 mM mannitol and 70 mM sucrose, and centrifuged at 1500×g for 5 min at 4℃, and the supernatants were collected for assay. The assay utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hapoxanthine. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. SOD activity was calculated and expressed as units per milliliter (U/mL) in plasma or units per milligram protein (U/mg protein) in tissue preparations.

In order to characterize subcellular distribution of targeted proteins, tissue samples were processed to isolate membrane and cytosolic fractions as previously described.26 Briefly, 150 mg of heart, lung, kidney and liver tissue samples were pulverized separately with mortar and pestle in liquid nitrogen, and then homogenized using a Polytron homogenizer in 1.5 mL of cold buffer A, which contained (in mM) 50 Tris-HCl (pH 7.5), 5 MgC1₂, 10 EGTA, 2 EDTA, 1 NaHCO₃, 1 PMSF, 1 β-glycerophosphate, 1 NaF, 1 Na₃VO₄, and (in µg/mL) 20 leupeptin and 4 aprotinin. The homogenate was centrifuged at 1000×g for 10 min at 4℃. The supernatant was collected and used as total tissue extracts. The supernatant was then further centrifuged at 100000×g for 60 min at 4℃. The supernatant was removed as cytosol fractions. The pellets were resuspended in buffer B containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM PMSF, 1 mM β-glycerophosphate, 60 mM Octyl β-glucopyranoside, 1 mM NaF, 0.1 mM Na₃VO₄, and (in µg/mL) 20 leupeptin and 4 aprotinin. The suspensions were homogenized again, held on ice for 30 min, and centrifuged at 100000×g for 60 min at 4℃. The supernatant was collected as membrane fractions. The proteins from each fraction (50-100 µg) were resolved via SDS-PAGE and subsequently transferred to PVDF membrane for immunoblot analysis. The membranes were blocked in 5% non fat milk for 2 h at room temperature, washed three times with TBS-T buffer each for 5 min, and then incubated overnight at 4℃ with primary antibodies raised against Cu/Zn-SOD (rabbit antiserum), Mn-SOD (rabbit polyclonal IgG) (1 : 1000, Millipore Corporation), p22phox and p67phox (rabbit polyclonal antibody, 1 : 500, Santa Cruz Biotechnology), respectively. After washing three times with TBS-T for 30 min, the blot was incubated for 1 h at room temperature with anti-rabbit IgG, HRP-linked antibody (1 : 10000, Cell Signaling). After washing the membranes, the blot was developed with enhanced chemiluminescence reagent (GE Healthcare) and then exposed to X-ray film. The same blot was stripped and reblotted with antibodies to ward GAPDH (1 : 2000, Cell Signaling) in total tissue extracts and cytosol fractions, or Na, K-ATPase (1 : 2000, Cell Signaling) in membrane fractions as an internal control. The intensity of the bands was quantified using image analysis software (Bio-Rad, Hercules, CA, USA), and the results were normalized to corresponding internal controls to correct for loading. Data are presented as percent change relative to the measurement in control rats.

The values are presented as mean±S.E.M. All biochemical assays were performed in duplicate and averaged before being analyzed. One-way or two-way analysis of variance was used for statistical analyses of data obtained within the same group and between groups, respectively. Tukey's test was used for multiple comparisons of group means. Significance was defined as p<0.05.

Administration of STZ resulted in characteristic symptoms of diabetes including hyperglycemia, and reduced body weight gain along with increased food and fluid intake when compared to age-matched controls (Table 1). Food consumption, water intake and plasma glucose levels in the diabetic group were much higher than those in the age-matched control group (p<0.01). NAC treatment significantly reduced water intake and food consumption compared with the diabetic untreated group (p<0.05), but did not significantly affect glucose levels. Body weight in the diabetic group was lower than that of the control group (p<0.05), and NAC treatment did not have significant effects on body weight gain (Table 1).

Diabetes is associated with disorders of many inflammatory cytokines.27 As shown in Table 2, the plasma levels of TNF-alpha and IL-6 in diabetic rats were increased significantly as compared to control rats (p<0.05), which were prevented by treatment with NAC.

Plasma (Fig. 1A) and tissue (Fig. 1B) levels of free 15-F_2t-IsoP in the diabetic group were much higher than those in the control group (all p<0.05 or p<0.01). NAC treatment significantly reduced free 15-F_2t-IsoP concentrations in the plasma and prevented the significant increase of 15-F_2t-IsoP in the heart, lung and liver tissues seen in the diabetic group (all p<0.05, D+NAC vs. D). NAC significantly reduced, but did not normalize 15-F_2t-IsoP levels in the kidney (p<0.05, D+NAC vs. D; p<0.05, D+NAC vs. C) (Fig. 1B).

Total antioxidant activity represents the total antioxidant capacity in tissues or in the plasma. Plasma total antioxidant activity in diabetic rats was higher than that of the control group (p<0.01) (Fig. 2A). NAC treatment significantly attenuated this increase in plasma total antioxidant levels in diabetic rats (p<0.05, D+NAC vs. D) (Fig. 2A). Changes in tissue total antioxidant activity and the effects of NAC treatment were tissue-specific. The total antioxidant activity in the heart in the diabetic group was higher than that in the control group (p<0.05) and was unaffected by NAC treatment (Fig. 2B). In contrast, however, total antioxidant activity in the lung, liver and kidney tissues was significantly decreased in the diabetic group as compared with control group (p<0.05) (Fig. 2B). NAC treatment elevated total antioxidant activity in the lung and liver (p<0.05, D+NAC vs. D), but not in the kidney.

The amount of SOD present in the cellular and extracellular environments is crucial for prevention of ROS mediated oxidative cellular damage. Plasma total SOD activity in the diabetic rats was much lower than that in the control group (p<0.01) (Fig. 3A), NAC did not have significant effects on plasma total SOD activity. In the control rats, tissue total SOD activities in the heart and lung were much lower than those in the liver and kidney (Fig. 3B), suggesting that SOD might not be the predominant endogenous antioxidant enzyme in the heart and lung in rats under physiological conditions. In the heart, total SOD activity was slightly increased in diabetic rats as compared to control rats (p<0.05, D vs. C), and this compensatory increase in SOD activity was not significantly affected by NAC treatment. While total SOD activity in the lung, was slightly decreased in diabetic rats as compared to control rats, NAC prevented the reduction of SOD activity in the lung. Furthermore, total SOD activities both in the liver and kidney were decreased significantly in diabetic rats as compared to age-matched controls (all p<0.05) (Fig. 3B), and NAC treatment prevented the decrease in total SOD activity in the liver, but not in the kidney.

In the heart and lung tissues, the levels of Cu/Zn-SOD protein in the diabetic group increased significantly compared with controls (all p<0.05, D vs. C) (Fig. 4A). NAC attenuated this increase in Cu/Zn-SOD protein expression in the heart (p< 0.05, D+NAC vs. D), but did not significantly affect Cu/Zn-SOD protein expression in the lung (Fig. 4A). In contrast, however, Cu/Zn-SOD protein expression in liver and kidney tissues in diabetic rats was lower than that of age-matched control rats (p<0.05 in liver, p<0.01 in kidney) (Fig. 4A). NAC prevented the decrease of Cu/Zn-SOD protein in the liver and attenuated its reduction in the kidney (all p<0.05, D+NAC vs. D) (Fig. 4A).

The changes of Mn-SOD protein expression in various tissues were different from those of Cu/Zn-SOD. There was no significant difference in Mn-SOD protein expression in the heart tissue among control, diabetic and NAC-treated diabetic groups (Fig. 4B). However, Mn-SOD protein expression in both the lung and the kidney decreased significantly in the diabetic group as compared to the control group, and NAC prevented the reduction of Mn-SOD protein in the lung (p<0.05, D+NAC vs. D) but further decreased its level in the kidney (p<0.05, D+NAC vs. D) (Fig. 4B). It is of an interest to note that Mn-SOD protein expression in the liver increased significantly in diabetic rats as compared control rats, but this increase was not affected by NAC treatment (Fig. 4B).

NADPH oxidase has been shown to be a major source of ROS production,10,28 and its subunits p22phox and p67phox are increased in the myocardium of STZ-induced diabetic rats.16 In the present study, we detected p22phox and p67phox in various tissues. As shown in Fig. 5A, p22phox protein levels in the heart, lung and kidney of the diabetic group were significantly higher than those of the control group, and NAC prevented the increase of p22phox protein expression in the lung, but did not normalize their increase in the heart and kidney (all p<0.05, D+NAC vs. D) (Fig. 5A). While there was no significant difference in p22phox protein expression in the liver between control and diabetic rats, 4 weeks of NAC treatment decreased its expression significantly (p<0.05, D+NAC vs. D or C) (Fig. 5A).

The p67phox protein expression was significantly increased both in the cytosol and membrane fractions in the lung, liver and kidney tissues of diabetic rats relative to control rats (Fig. 5B and C), reflecting increased p67phox activation as well as increased total protein levels which were in line with the increases of total p67phox protein expression in these tissues (data not shown). However, p67phox protein expression in the heart was significantly increased in the membrane fractions while markedly decreased in the cytosol fractions, reflecting increased p67phox activation. NAC prevented the increase of p67phox membrane translocation in the heart and liver and significantly attenuated the increase of p67phox membrane translocation in the lung and kidney tissues (all p<0.05, D+NAC vs. D) (Fig. 5C).