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Experimental data suggesting that inflammation mediated rat liver mitochondrial dysfunction results from secondary hypoxia rather than from direct effects of inflammatory mediators.

Weidinger A, Dungel P, Perlinger M, Singer K, Ghebes C, Duvigneau JC, Müllebner A, Schäfer U, Redl H, Kozlov AV - Front Physiol (2013)

Bottom Line: To eliminate this interaction, precision cut liver slices (PCLS) were used in this study aiming to dissect the effects of HOX and IM on mitochondrial function, integrity of cellular membrane, and the expression of genes associated with inflammation.Elevated expression of interleukin 6 (IL-6) was found in both models reflecting converging pathways regulating the expression of this gene.Both models caused damage to hepatocytes resulting in the release of alanine aminotransferase (ALT).

View Article: PubMed Central - PubMed

Affiliation: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology Vienna, Austria.

ABSTRACT
Systemic inflammatory response (SIR) comprises both direct effects of inflammatory mediators (IM) and indirect effects, such as secondary circulatory failure which results in tissue hypoxia (HOX). These two key components, SIR and HOX, cause multiple organ failure (MOF). Since HOX and IM occur and interact simultaneously in vivo, it is difficult to clarify their individual pathological impact. To eliminate this interaction, precision cut liver slices (PCLS) were used in this study aiming to dissect the effects of HOX and IM on mitochondrial function, integrity of cellular membrane, and the expression of genes associated with inflammation. HOX was induced by incubating PCLS or rat liver mitochondria at pO2 < 1% followed by reoxygenation (HOX/ROX model). Inflammatory injury was stimulated by incubating PCLS with IM (IM model). We found upregulation of inducible nitric oxide synthase (iNOS) expression only in the IM model, while heme oxygenase 1 (HO-1) expression was upregulated only in the HOX/ROX model. Elevated expression of interleukin 6 (IL-6) was found in both models reflecting converging pathways regulating the expression of this gene. Both models caused damage to hepatocytes resulting in the release of alanine aminotransferase (ALT). The leakage of aspartate aminotransferase (AST) was observed only during the hypoxic phase in the HOX/ROX model. The ROX phase of HOX, but not IM, drastically impaired mitochondrial electron supply via complex I and II. Additional experiments performed with isolated mitochondria showed that free iron, released during HOX, is likely a key prerequisite of mitochondrial dysfunction induced during the ROX phase. Our data suggests that mitochondrial dysfunction, previously observed in in vivo SIR-models, is the result of secondary circulatory failure inducing HOX rather than the result of a direct interaction of IM with liver cells.

No MeSH data available.


Related in: MedlinePlus

Effect of IM (A,C) and HOX/ROX (B,D) on the release of ALT (A,B) and AST (C,D) from PCLS. (A,C) PCLS were incubated in NM only or NM containing LPS or LPS+IM at 37°C. ALT/AST samples were taken after 4 h incubation. (B,D) PCLS were incubated in NM which was either equilibrated with air (NOX, ROX) or nitrogen (HOX). The medium for ALT/AST analysis was taken at the end of the hypoxic phase (1 h) and the end of the subsequent reoxygenation phase (1 h HOX + 1 h ROX). Data are expressed as mean ± SEM of at least n = 4 and as a percentage of control. **p < 0.01; ***p < 0.001. Abbreviations used: ALT, alanine aminotransferase; AST, aspartate aminotransferase; NM, normal medium; LPS, lipopolysaccharide; IM, inflammatory mediators; NOX, Normoxia; HOX, Hypoxia; ROX, reoxygenation; PCLS, precision cut liver slices.
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Figure 1: Effect of IM (A,C) and HOX/ROX (B,D) on the release of ALT (A,B) and AST (C,D) from PCLS. (A,C) PCLS were incubated in NM only or NM containing LPS or LPS+IM at 37°C. ALT/AST samples were taken after 4 h incubation. (B,D) PCLS were incubated in NM which was either equilibrated with air (NOX, ROX) or nitrogen (HOX). The medium for ALT/AST analysis was taken at the end of the hypoxic phase (1 h) and the end of the subsequent reoxygenation phase (1 h HOX + 1 h ROX). Data are expressed as mean ± SEM of at least n = 4 and as a percentage of control. **p < 0.01; ***p < 0.001. Abbreviations used: ALT, alanine aminotransferase; AST, aspartate aminotransferase; NM, normal medium; LPS, lipopolysaccharide; IM, inflammatory mediators; NOX, Normoxia; HOX, Hypoxia; ROX, reoxygenation; PCLS, precision cut liver slices.

Mentions: Initially the experimental HOX/ROX-PCLS and IM-PCLS models were standardized. Standardization of the models is based on a significant elevation of ALT release as a basic marker of hepatocyte damage. In the IM-PCLS model significantly elevated ALT levels were observed following 4 h of incubation with IM (Figure 1A), while AST levels remained unchanged (Figure 1C). In the HOX/ROX-PCLS model pronounced and statistically significantly elevated ALT and AST levels were observed after 1 h of hypoxia. No further release of ALT or AST was observed after subsequent ROX (1 h) (Figures 1B,D). To analyze whether IM induced inflammatory response in PCLS, we tested the expression of genes associated with inflammation.


Experimental data suggesting that inflammation mediated rat liver mitochondrial dysfunction results from secondary hypoxia rather than from direct effects of inflammatory mediators.

Weidinger A, Dungel P, Perlinger M, Singer K, Ghebes C, Duvigneau JC, Müllebner A, Schäfer U, Redl H, Kozlov AV - Front Physiol (2013)

Effect of IM (A,C) and HOX/ROX (B,D) on the release of ALT (A,B) and AST (C,D) from PCLS. (A,C) PCLS were incubated in NM only or NM containing LPS or LPS+IM at 37°C. ALT/AST samples were taken after 4 h incubation. (B,D) PCLS were incubated in NM which was either equilibrated with air (NOX, ROX) or nitrogen (HOX). The medium for ALT/AST analysis was taken at the end of the hypoxic phase (1 h) and the end of the subsequent reoxygenation phase (1 h HOX + 1 h ROX). Data are expressed as mean ± SEM of at least n = 4 and as a percentage of control. **p < 0.01; ***p < 0.001. Abbreviations used: ALT, alanine aminotransferase; AST, aspartate aminotransferase; NM, normal medium; LPS, lipopolysaccharide; IM, inflammatory mediators; NOX, Normoxia; HOX, Hypoxia; ROX, reoxygenation; PCLS, precision cut liver slices.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675332&req=5

Figure 1: Effect of IM (A,C) and HOX/ROX (B,D) on the release of ALT (A,B) and AST (C,D) from PCLS. (A,C) PCLS were incubated in NM only or NM containing LPS or LPS+IM at 37°C. ALT/AST samples were taken after 4 h incubation. (B,D) PCLS were incubated in NM which was either equilibrated with air (NOX, ROX) or nitrogen (HOX). The medium for ALT/AST analysis was taken at the end of the hypoxic phase (1 h) and the end of the subsequent reoxygenation phase (1 h HOX + 1 h ROX). Data are expressed as mean ± SEM of at least n = 4 and as a percentage of control. **p < 0.01; ***p < 0.001. Abbreviations used: ALT, alanine aminotransferase; AST, aspartate aminotransferase; NM, normal medium; LPS, lipopolysaccharide; IM, inflammatory mediators; NOX, Normoxia; HOX, Hypoxia; ROX, reoxygenation; PCLS, precision cut liver slices.
Mentions: Initially the experimental HOX/ROX-PCLS and IM-PCLS models were standardized. Standardization of the models is based on a significant elevation of ALT release as a basic marker of hepatocyte damage. In the IM-PCLS model significantly elevated ALT levels were observed following 4 h of incubation with IM (Figure 1A), while AST levels remained unchanged (Figure 1C). In the HOX/ROX-PCLS model pronounced and statistically significantly elevated ALT and AST levels were observed after 1 h of hypoxia. No further release of ALT or AST was observed after subsequent ROX (1 h) (Figures 1B,D). To analyze whether IM induced inflammatory response in PCLS, we tested the expression of genes associated with inflammation.

Bottom Line: To eliminate this interaction, precision cut liver slices (PCLS) were used in this study aiming to dissect the effects of HOX and IM on mitochondrial function, integrity of cellular membrane, and the expression of genes associated with inflammation.Elevated expression of interleukin 6 (IL-6) was found in both models reflecting converging pathways regulating the expression of this gene.Both models caused damage to hepatocytes resulting in the release of alanine aminotransferase (ALT).

View Article: PubMed Central - PubMed

Affiliation: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology Vienna, Austria.

ABSTRACT
Systemic inflammatory response (SIR) comprises both direct effects of inflammatory mediators (IM) and indirect effects, such as secondary circulatory failure which results in tissue hypoxia (HOX). These two key components, SIR and HOX, cause multiple organ failure (MOF). Since HOX and IM occur and interact simultaneously in vivo, it is difficult to clarify their individual pathological impact. To eliminate this interaction, precision cut liver slices (PCLS) were used in this study aiming to dissect the effects of HOX and IM on mitochondrial function, integrity of cellular membrane, and the expression of genes associated with inflammation. HOX was induced by incubating PCLS or rat liver mitochondria at pO2 < 1% followed by reoxygenation (HOX/ROX model). Inflammatory injury was stimulated by incubating PCLS with IM (IM model). We found upregulation of inducible nitric oxide synthase (iNOS) expression only in the IM model, while heme oxygenase 1 (HO-1) expression was upregulated only in the HOX/ROX model. Elevated expression of interleukin 6 (IL-6) was found in both models reflecting converging pathways regulating the expression of this gene. Both models caused damage to hepatocytes resulting in the release of alanine aminotransferase (ALT). The leakage of aspartate aminotransferase (AST) was observed only during the hypoxic phase in the HOX/ROX model. The ROX phase of HOX, but not IM, drastically impaired mitochondrial electron supply via complex I and II. Additional experiments performed with isolated mitochondria showed that free iron, released during HOX, is likely a key prerequisite of mitochondrial dysfunction induced during the ROX phase. Our data suggests that mitochondrial dysfunction, previously observed in in vivo SIR-models, is the result of secondary circulatory failure inducing HOX rather than the result of a direct interaction of IM with liver cells.

No MeSH data available.


Related in: MedlinePlus