Endocrinol Metab.  2014 Sep;29(3):356-362. 10.3803/EnM.2014.29.3.356.

Protective Effects of Inducible HO-1 on Oxygen Toxicity in Rat Brain Endothelial Microvessel Cells

• Yoo SJ
• Nakra NK
• Ronnett GV
• Moon C

• KMID: 2169473
• DOI: http://doi.org/10.3803/EnM.2014.29.3.356

Abstract

BACKGROUND
Reperfusion in ischemia is believed to generate cytotoxic oxidative stress, which mediates reperfusion injury. These stress conditions can initiate lipid peroxidation and damage to proteins, as well as promote DNA strand breaks. As biliverdin and bilirubin produced by heme oxygenase isoform 1 (HO-1) have antioxidant properties, the production of both antioxidants by HO-1 may help increase the resistance of the ischemic brain to oxidative stress. In the present study, the survival effect of HO-1 was confirmed using hemin.
METHODS
To confirm the roles of HO-1, carbon monoxide, and cyclic guanosine monophosphate further in the antioxidant effect of HO-1 and bilirubin, cells were treated with cycloheximide, desferoxamine, and zinc deuteroporphyrin IX 2,4 bis glycol, respectively.
RESULTS
HO-1 itself acted as an antioxidant. Furthermore, iron, rather than carbon monoxide, was involved in the HO-1-mediated survival effect. HO-1 activity was also important in providing bilirubin as an antioxidant.
CONCLUSION
Our results suggested that HO-1 helped to increase the resistance of the ischemic brain to oxidative stress.

Keyword

Heme; Oxygenases; Bilirubin; Iron; Carbon monoxide

MeSH Terms

Animals
Antioxidants
Bilirubin
Biliverdine
Brain*
Carbon Monoxide
Cycloheximide
DNA
Guanosine Monophosphate
Heme
Heme Oxygenase (Decyclizing)
Hemin
Iron
Ischemia
Lipid Peroxidation
Microvessels*
Oxidative Stress
Oxygen*
Oxygenases
Rats*
Reperfusion
Reperfusion Injury
Zinc
Antioxidants
Bilirubin
Biliverdine
Carbon Monoxide
Cycloheximide
DNA
Guanosine Monophosphate
Heme
Heme Oxygenase (Decyclizing)
Hemin
Iron
Oxygen
Oxygenases
Zinc

Figure

• 

  Fig. 1 Protective effects of heme under conditions of oxidative stress. (A) Experimental scheme for induction, sensitization, and oxidative stress. The "H (0.5) H (10)" condition was induction by hemin (0.5 µM) and sensitization by hemin (10 µM), while the "none" condition was no induction and no sensitization. (B) Cells untreated by H2O2 were used as a positive control. Before oxidative stress was applied (500 µM H2O2), cells were inducted and sensitized according to the experimental scheme. Data are mean±SEM of three separate experiments. Analyses performed were one-way analysis of variance followed by Dunnett's post hoc test. aP<0.01 denote statistical significance.

• 

  Fig. 2 The antioxidant effects of heme oxygenase isoform 1 activity require iron. (A) Experimental scheme for induction, sensitization, and oxidative stress. The "C (20) H (10)," "D (5) H (10)," and "Z (20) H (10)" conditions were induction by cycloheximide (20 ng/mL), desferoxamine (5 nM), and ZnBG (20 µM), respectively, with sensitization by hemin (10 µM) in each case, while the "none" condition was no induction and no sensitization. (B) Cells untreated by H2O2 were used as a positive control. Before oxidative stress was applied (500 µM H2O2), cells were inducted and sensitized according to the experimental scheme. Data are mean±SEM of three separate experiments. Analyses performed were one-way analysis of variance followed by Dunnett's post hoc test. aP<0.01 denote statistical significance.

• 

  Fig. 3 The antioxidant effects of bilirubin require heme oxygenase isoform 1 activity. (A) Experimental scheme for induction, sensitization, and oxidative stress. The "C (20) B (10)," "D (5) B (10)," and "Z (20) B (10)" conditions were induction by cycloheximide (20 ng/mL), desferoxamine (5 nM), and ZnBG (20 µM), respectively, with sensitization by bilirubin (1 µM) in each case, while the "none" condition was no induction and no sensitization. (B) Cells untreated by H2O2 were used as a positive control. Before oxidative stress was applied (500 µM H2O2), cells were inducted and sensitized according to the experimental scheme. Data are mean±SEM of three separate experiments. Analyses performed were one-way analysis of variance followed by Dunnett's post hoc test. aP<0.001 denote statistical significance.

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