Abstract
Background Organ injury and dysfunction in sepsis accounts for significant morbidity and mortality. Adaptive cellular responses in the setting of sepsis prevent injury and allow for organ recovery. We and others have shown that part of the adaptive response includes regulation of cellular respiration and maintenance of a healthy mitochondrial population. Herein, we hypothesized that endotoxin-induced changes in hepatocyte mitochondrial respiration and homeostasis are regulated by an inducible nitric oxide synthase/nitric oxide (iNOS/NO)-mitochondrial reactive oxygen species (mtROS) signaling axis, involving activation of the NRF2 signaling pathway. Methods Wild-type (C57Bl/6) or iNos−/− male mice were subjected to intraperitoneal lipopolysaccharide (LPS) injections to simulate endotoxemia. Individual mice were randomized to treatment with NO-releasing agent DPTA-NONOate, mtROS scavenger MitoTEMPO, or vehicle controls. Other mice were treated with scramble or Nrf2-specific siRNA via tail vein injection. Primary murine hepatocytes were utilized for in vitro studies with or without LPS stimulation. Oxygen consumption rates were measured to establish mitochondrial respiratory parameters. Western blotting, confocal microscopy with immunocytochemistry, and rtPCR were performed for analysis of iNOS as well as markers of both autophagy and mitochondrial biogenesis. Results LPS treatment inhibited aerobic respiration in vitro in wild-type but not iNos−/− cells. Experimental endotoxemia in vivo or in vitro induced iNOS protein and mtROS production. However, induction of mtROS was dependent on iNOS expression. Furthermore, LPS-induced hepatic autophagy/mitophagy and mitochondrial biogenesis were significantly attenuated in iNos−/− mice or cells with NO or mtROS scavenging. These responses were rescued in iNos−/− mice via delivery of NO both in vivo and in vitro. Conclusions. These data suggest that regulation of mitochondrial quality control following hepatocyte LPS exposure is dependent at least in part on a NO-mtROS signaling network. Further investigation to identify specific agents that modulate this process may facilitate the prevention of organ injury in sepsis.
Highlights
Severe sepsis represents a major healthcare burden in the United States with annual case incidence exceeding 750,000 and an associated mortality of nearly 30% [1]
The present data demonstrate that LPS-associated reduction in the rate of oxygen consumption in hepatocytes was dependent on iNOS expression (Figure 1(c))
MitoTEMPO was able to prevent appropriate induction of proliferator-activated receptor gamma coactivator 1-α (PGC-1α) and LC3 protein following LPS exposure as measured by immunocytochemistry in liver sections from treated mice (Figure 4(c)). Taken together these data show that iNOS is upregulated on exposure to LPS, stimulating increased mitochondrial biogenesis and autophagy in an mitochondrial reactive oxygen species (mtROS)-Nuclear factor erythroid 2-related factor 2 (NRF2)-dependent manner
Summary
Severe sepsis represents a major healthcare burden in the United States with annual case incidence exceeding 750,000 and an associated mortality of nearly 30% [1]. In a rat model of peritonitis, the severity of organ dysfunction was found to correlate with the degree of mitochondrial dysfunction and the overproduction of nitric oxide (NO) [6] These findings implicate the mitochondria as important mediators of the clinical consequences of sepsis [7]. LPS-induced hepatic autophagy/mitophagy and mitochondrial biogenesis were significantly attenuated in iNos-/- mice or cells with NO or mtROS scavenging. These responses were rescued in iNos-/- mice via delivery of NO both in vivo and in vitro. Further investigation to identify specific agents that modulate this process may facilitate the prevention of organ injury in sepsis
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