Mitochondrial anti-oxidant protects IEX-1 deficient mice from organ damage during endotoxemia

Abstract

Sepsis, a leading cause of mortality in intensive care units worldwide, is often a result of overactive and systemic inflammation following serious infections. We found that mice lacking immediate early responsive gene X-1 (IEX-1) were prone to lipopolysaccharide (LPS) -induced endotoxemia. A nonlethal dose of LPS provoked numerous aberrations in IEX-1 knockout (KO) mice including pancytopenia, increased serum aspartate aminotransferase (AST), and lung neutrophilia, concurrent with liver and kidney damage, followed by death. Given these results, in conjunction with a proven role for IEX-1 in the regulation of reactive oxygen species (ROS) homeostasis during stress, we pre-treated IEX-1 KO mice with Mitoquinone (MitoQ), a mitochondrion-based antioxidant prior to LPS injection. The treatment significantly reduced ROS formation in circulatory cells and protected against pancytopenia and multiple organ failure, drastically increasing the survival rate of IEX-1 KO mice challenged by this low dose of LPS. This study confirms significant contribution of mitochondrial ROS to the etiology of sepsis.

Figure 1

IEX-1 deficient mice are prone to LPS-induced endotoxemia. Kaplan-Meyer analysis of the survival of WT and KO mice subjected to a nonlethal dose of LPS is shown. Data shown are from two experiments with n= 8 in each treatment group.

Figure 2

MitoQ pre-treatment reverses endotoxemic effects of LPS in IEX-1 deficient mice. Kaplan-Meyer analysis is shown for the survival of WT and KO mice with MitoQ pre-treatment before LPS challenge as in Figure 1(A) with n= 8 in each WT and KO treatment group. Platelet (B) and red blood cell (C) levels were measured, alongside red blood cell mean corpuscular volume (D) and distribution width (E). Changes in red blood cell morphology were visible through blood representative smears (100x magnification) (F). Granulocyte levels were also noted (G). Data shown (B–E, G) are from two experiments with n= 5 in each treatment group. Groups are denoted as Control (white), LPS (black), and LPS+ MitoQ (grey). *P< 0.05 and **P< 0.01

Figure 3

LPS induced lung neutrophilia and tissue damage are exacerbated in the loss of IEX-1. Representative H&E stainings of lung tissues were prepared from WT and KO control mice, or with LPS and MitoQ+LPS treatments (A). Bar (100μm). PMN were counted in the lung tissue of subject mice (B). Groups are denoted as Control (white), LPS (black), and LPS+ MitoQ (grey). Data shown are from two experiments where n= 5. **P< 0.01

Figure 4

MitoQ protects from LPS induced kidney and liver damage. Kidneys (A) and livers (B) of control and treated WT and KO mice were harvested 24 hours after LPS injection. Serum AST was measured in all subjects; Control (white), LPS (black), and LPS+ MitoQ (grey)(C). H&E stains are representative of two experiments with n= 5. Bar (100μm). Serum AST is a result of two experiments with n=3. *P< 0.05

Figure 5

Analysis of ROS and MMP in circulating blood cells. Cellular levels of ROS were analyzed in granulocyte (A), red blood cell (B) and platelet (C) populations from 2 experiments where n=5. JC-1 aggregation ratios are expressed as a ratio of red to green fluorescence of JC-1 in platelets collected from mice at a standing state, or MitoQ treated and nontreated groups 24 hours post-endotoxemic challenge (D). Data shown is from 2 experiments with similar results and n= 3 in each. Groups are denoted as Control (white), LPS (black), and LPS+ MitoQ (grey). *P< 0.05 and **P< 0.01