Effects of Short Term Antioxidant Cocktail Supplementation on the Oxidative Stress and Inflammatory Response of Renal Inflammation in Diabetic Mice

Seul-Ki Park; Na-Young Park; Yunsook Lim

doi:10.4163/kjn.2009.42.8.673

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Park, Park, and Lim: Effects of Short Term Antioxidant Cocktail Supplementation on the Oxidative Stress and Inflammatory Response of Renal Inflammation in Diabetic Mice

Original Article

Korean Journal of Nutrition

Korean Journal of Nutrition 2009; 42(8): 673-681.

Published online: 31 December 2009

DOI: https://doi.org/10.4163/kjn.2009.42.8.673

Effects of Short Term Antioxidant Cocktail Supplementation on the Oxidative Stress and Inflammatory Response of Renal Inflammation in Diabetic Mice

Seul-Ki Park, Na-Young Park, Yunsook Lim

Department of Food and Nutrition, Research Institute of Human Ecology, Kyunghee University, Seoul 130-701, Korea.

To whom correspondence should be addressed. (ylim@khu.ac.kr)

Received 18 August 2009 Revised 26 October 2009 Accepted 1 December 2009

Abstract

Diabetes mellitus is a multifactorial disease. Particularly, diabetic nephropathy is a serious complication for diabetic patients, yet the precise mechanisms that underline the initial stage of diabetic renal inflammation remain unknown. However, oxidative stress induced by hyperglycemia in diabetes is implicated in diabetic renal disease. We hypothesized that dietary supplementation of antioxidants either VCE (0.5% VC + 0.5% VE) or Comb (0.5% VC + 0.5% VE + 2.5% N-acetylcysteine) improves acute diabetic renal inflammation through modulation of blood glucose levels and antioxidant and anti-inflammatory responses. Experimental animals (5.5 weeks old female ICR) used were treated with alloxan (180 mg/kg) once. When fasting blood glucose levels were higher than 250 mg/dL, mice were divided into 3 groups fed different levels of antioxidant supplementation, DM (diabetic mice fed AIN 93G purified rodent diet); VCE (diabetic mice fed 0.5% vitamin C and 0.5% vitamin E supplemented diet); Comb (diabetic mice fed 0.5% vitamin C, 0.5% vitamin E and 2.5% N-acetylcysteine supplemented diet), for 10 days and then sacrificed. Body weights were measured once a week and blood glucose levels were monitored twice a week. Lipid peroxidation products, thiobarbituric acid reacting substances were measured in kidney. NF-κB activation was indirectly demonstrated by pIκB-α and expressions of selective inflammatory and oxidative stress markers including antioxidant enzymes were also determined. Dietary antioxidant supplementation improved levels of blood glucose as well as kidney lipid peroxi-dation. Dietary antioxidant supplementation improved NF-κB activation and protein expression of HO-1, but not mRNA expression levels in diabetic mice fed Comb diet. In contrast, the mRNA and protein expression of CuZnSOD was decreased in diabetic mice fed Comb diet. However, antioxidant supplementation did not improve mRNA and protein expressions of IL-1β and MnSOD in diabetic mice. These findings demonstrate that acute diabetic renal inflammation was associated with altered inflammatory and antioxidant responses and suggest that antioxidant cocktail supplementation may have beneficial effects on early stage of diabetic nephropathy through modulation of blood glucose levels and antioxidant enzyme expressions.

Keywords: diabetic nephropathy, antioxidant, oxidative stress, inflammation, diabetes mellitus

Figures and Tables

Fig. 1

Dietary antioxidant supplementation reduces blood glucose levels in alloxan induced diabetic mice. Mice were injected with alloxan (180 mg/kg). DM (diabetes mellitus), VCE (0.5% Vit C and 0.5% Vit E), Comb (0.5% Vit C and 0.5% Vit E and 2.5% NAC) Values are means ± SEM. Means for a variable without a common letter differ. p < 0.05.

Fig. 2

Dietary antioxidant supplementation reduces renal lipid peroxidation in alloxan induced diabetic mice. DM (Diabetes mellitus), VCE (0.5% Vit C and 0.5% Vit E), Comb (0.5% Vit C and 0.5% Vit E and 2.5% NAC) Values are means ± SEM. Means for a variable without a common letter differ. p < 0.05.

Fig. 3

Effects of antioxidant supplementation on mRNA expression levels of oxidative stress markers in diabetic kidney. DM (diabetes mellitus), VCE (0.5% vit C and 0.5% Vit E), Comb (0.5% vit C and 0.5% vit E and 2.5% NAC) Values are means ± SEM. Means for a variable without a common letter differ. p < 0.05. A: IkB-α (IkappaB-alpha). B: MnSoD (maganase superoxide dismutase). C: CuZnSOD (copper zinc superoxide dismutase).

Fig. 4

Effects of antioxidant supplementation on protein expression levels of oxidative stress markers and pro-inflammatory cytokines in diabetic kidney. DM (diabetes mellitus), VCE (0.5% vit C and 0.5% Vit E), Comb (0.5% vit C and 0.5% vit E and 2.5% NAC) Values are means ± SEM. Means for a variable without a common letter differ. p < 0.05. A: p-IκB-α (phosphorylated-Ikappaβ-alpha). B: IL-1β (interleukin-1 beta). C: HO-1 (heme oxygenase-1). D: MnSOD (maganase superoxide dismutase). E: CuZnSOD (copper zinc superoxide dismutase).

Table 1

Composition of experimental diets

1) Vitamin C: L-ASCORBIC ACID, > = 99.0%, CRYSTALLINE, Sigma-Aldrich St. Louis, MO., USA

2) Vitamin E: (+/-)-ALPHA-TOCOPHEROL, SYNTHETIC, > = 96%, Sigma-Aldrich, St. Louis, MO., USA

3) NAC: N-ACETYL-L-CYSTEINE, SIGMA GRADE, > = 99%, Sigma-Aldrich, St. Louis, MO., USA

Table 2

Reverse transcriptase PCR primer sequences

Notes

This research was supported by grants from Korea Research Foundation (KRF-2007-028-C00278).

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