Yonsei Med J.  2012 Mar;53(2):294-303. 10.3349/ymj.2012.53.2.294.

Effects of N-Acetylcysteine on Nicotinamide Dinucleotide Phosphate Oxidase Activation and Antioxidant Status in Heart, Lung, Liver and Kidney in Streptozotocin-Induced Diabetic Rats

• Lei S
• Liu Y
• Liu H
• Yu H
• Wang H
• Xia Z

• KMID: 1120195
• DOI: http://doi.org/10.3349/ymj.2012.53.2.294

Abstract

PURPOSE
Hyperglycemia increases reactive oxygen species (ROS) and the resulting oxidative stress plays a key role in the pathogenesis of diabetic complications. Nicotinamide dinucleotide phosphate (NADPH) oxidase is one of the major sources of ROS production in diabetes. We, therefore, examined the possibility that NADPH oxidase activation is increased in various tissues, and that the antioxidant N-acetylcysteine (NAC) may have tissue specific effects on NADPH oxidase and tissue antioxidant status in diabetes.
MATERIALS AND METHODS
Control (C) and streptozotocin-induced diabetic (D) rats were treated either with NAC (1.5 g/kg/day) orally or placebo for 4 weeks. The plasma, heart, lung, liver, kidney were harvested immediately and stored for biochemical or immunoblot analysis.
RESULTS
levels of free 15-F2t-isoprostane were increased in plasma, heart, lung, liver and kidney tissues in diabetic rats, accompanied with significantly increased membrane translocation of the NADPH oxidase subunit p67phox in all tissues and increased expression of the membrane-bound subunit p22phox in heart, lung and kidney. The tissue antioxidant activity in lung, liver and kidney was decreased in diabetic rats, while it was increased in heart tissue. NAC reduced the expression of p22phox and p67phox, suppressed p67phox membrane translocation, and reduced free 15-F(2t)-isoprostane levels in all tissues. NAC increased antioxidant activity in liver and lung, but did not significantly affect antioxidant activity in heart and kidney.
CONCLUSION
The current study shows that NAC inhibits NADPH oxidase activation in diabetes and attenuates tissue oxidative damage in all organs, even though its effects on antioxidant activity are tissue specific.

Keyword

Antioxidant status; diabetes; 15-F2t-isoprostane; N-acetylcysteine; NADPH oxidase

MeSH Terms

Acetylcysteine/*therapeutic use
Animals
Antioxidants/*metabolism
Diabetes Mellitus, Experimental/*drug therapy/*metabolism
Heart/drug effects
Kidney/drug effects/metabolism
Liver/drug effects/metabolism
Lung/drug effects/metabolism
Male
NADPH Oxidase/*metabolism
Rats
Rats, Sprague-Dawley

Figure

• 

  Fig. 1 Effects of N-acetylcysteine treatment on the level of free 15-F2t-isoprostane in plasma (A) and various tissues (B). Control (C) or STZ-induced diabetic rats were either untreated (D) or treated with the antioxidant N-acetylcysteine (1.5 g/kg/day, D+NAC) by oral gavage for four weeks. Results are expressed as means±S.E.M., n=7, *p<0.05, **p<0.01 vs. C; †p<0.05, ‡p<0.01 vs. D. STZ, streptozotocin.

• 

  Fig. 2 Effects of N-acetylcysteine treatment on the level of total antioxidant activity in plasma (A) and various tissues (B). Control (C) or STZ-induced diabetic rats were either untreated (D) or treated with the antioxidant N-acetylcysteine (1.5 g/kg/day, D+NAC) by oral gavage for four weeks. Results are expressed as means±S.E.M., n=7, *p<0.05, **p<0.01 vs. C; †p<0.05 vs. D. STZ, streptozotocin.

• 

  Fig. 3 Effects of N-acetylcysteine treatment on the level of total SOD activity in plasma (A) and various tissues (B). Control (C) or STZ-induced diabetic rats were either untreated (D) or treated with the antioxidant N-acetylcysteine (1.5 g/kg/day, D+NAC) by oral gavage for four weeks. Results are expressed as means±S.E.M., n=7 per group, *p<0.05, **p<0.01 vs. C; †p<0.05 vs. D. SOD, superoxide dismutase; STZ, streptozotocin.

• 

  Fig. 4 Western blots analysis of Cu/Zn-SOD and Mn-SOD protein expression in various tissues. Control (C) or STZ-induced diabetic rats were either untreated (D) or treated with the antioxidant N-acetylcysteine (1.5 g/kg/day, D+NAC) by oral gavage for four weeks. (A) (top) Representative Western blot showing Cu/Zn-SOD expression with GAPDH as a loading control in total tissue extracts; (bottom, graph) Cu/Zn-SOD densitometric values were normalized to their corresponding GAPDH densitometric values and expressed as percent change relative to the measurement in control rats. (B) (top) Representative Western blot showing Mn-SOD expression with GAPDH as a loading control in total tissue extracts; (bottom, graph) Mn-SOD densitometric values were normalized to their corresponding GAPDH densitometric values and expressed as percent change relative to the measurement in control rats. All the results are expressed as means±S.E.M., n=7, *p<0.05, **p<0.01 vs. C; †p<0.05 vs. D. SOD, superoxide dismutase; STZ, streptozotocin.

• 

  Fig. 5 Western blots analysis of NADPH oxidase subunits p22phox and p67phox protein expression in various tissues. Control (C) or STZ-induced diabetic rats were either untreated (D) or treated with the antioxidant N-acetylcysteine (1.5 g/kg/day, D+NAC) by oral gavage for four weeks. (A) (top) Representative Western blot showing p22phox expression with GAPDH as a loading control in total tissue extracts; (bottom, graph) p22phox densitometric values were normalized to their corresponding GAPDH densitometric values and expressed as percent change relative to the measurement in control rats. (B) (top) Representative Western blot showing p67phox expression with GAPDH as a loading control in cytosol fractions; (bottom, graph) p67phox densitometric values were normalized to their corresponding GAPDH densitometric values and expressed as percent change relative to the measurement in control rats. (C)(top) Representative Western blot showing p67phox expression with Na, K-ATPase as a loading control in membrane fractions; (bottom, graph) p67phox densitometric values were normalized to their corresponding Na, K-ATPase densitometric values and expressed as percent change relative to the measurement in control rats. All the results are expressed as means±S.E.M., n=7, *p<0.05, **p<0.01 vs. C; †p<0.05 vs. D. NADPH, nicotinamide dinucleotide phosphate; STZ, streptozotocin.

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