MKK3 depletion attenuates intestinal injury after traumatic hemorrhagic shock by restoring mitochondrial function.

BACKGROUND: Traumatic hemorrhagic shock (THS) is a complex pathophysiological process resulting in multiple organ failure. Intestinal barrier dysfunction is one of the mechanisms implicated in multiple organ failure. The present study aimed to explore the regulatory role of mitogen-activated protein kinase kinase 3 (MKK3) in THS-induced intestinal injury and to elucidate its potential mechanism. METHODS: Rats were subjected to trauma and hemorrhage to establish a THS animal model. MKK3-targeted lentiviral vectors were injected via the tail vein 72 h before modeling. Twelve hours post-modeling, the mean arterial pressure (MAP) and heart rate (HR) were monitored, and histological injury to the intestine was assessed via H&E staining and transmission electron microscopy. Mitochondrial function and mitochondrial reactive oxygen species (ROS) were evaluated. IEC-6 cells were exposed to hypoxia to mimic intestinal injury following THS in vitro. RESULTS: MKK3 deficiency alleviated intestinal injury and restored mitochondrial function in intestinal tissues from THS-induced rats and hypoxia-treated IEC-6 cells. In addition, MKK3 deficiency promoted Sirt1/PGC-1α-mediated mitochondrial biogenesis and restricted Pink1/Parkin-mediated mitophagy in the injured intestine and IEC-6 cells. Furthermore, the protective effect of MKK3 knockdown against hypoxia-induced mitochondrial damage was strengthened upon simultaneous LC3B/Pink1/Parkin knockdown or weakened upon simultaneous Sirt1 knockdown. CONCLUSION: MKK3 deficiency protected against intestinal injury induced by THS by promoting mitochondrial biogenesis and restricting excessive mitophagy.

Trauma-hemorrhagic shock induces a CD36-dependent RBC endothelial-adhesive phenotype.

OBJECTIVE: Microvascular dysfunction is a key element in the development of the multiple organ dysfunction syndrome. Although the mechanisms for this response are unclear, RBC adhesion to endothelium may initiate intravascular occlusion leading to ischemic tissue injury. Thus, we tested the hypothesis that trauma-hemorrhage induces RBC-endothelial cell adhesion. DESIGN: Prospective in vivo and in vitro animal study and analysis of patient blood samples. SETTING: University research laboratory and hospital emergency and trauma units. INTERVENTION: We initially assayed RBC adhesion to endothelial cells in vitro using RBCs obtained from rats subjected to trauma-hemorrhagic shock or sham shock as well as from severely injured trauma patients. Subsequently, we measured the role of putative RBCs and endothelial cell receptors in the increased RBC-endothelial cell adhesive response. MAIN RESULTS: In both rats and humans, trauma-hemorrhagic shock increased RBC adhesion to endothelium as well as increasing several putative RBC surface adhesion molecules including CD36. The critical factor leading to RBC-endothelial cell adhesion was increased surface RBC CD36 expression. Adhesion of trauma-hemorrhagic shock RBCs was mediated, at least in part, by the binding of RBC CD36 to its cognate endothelial receptors (αVß3 and VCAM-1). Gut-derived factors carried in the intestinal lymphatics triggered these trauma-hemorrhagic shock-induced RBC changes because 1) preventing trauma-hemorrhagic shock intestinal lymph from reaching the systemic circulation abrogated the RBC effects, 2) in vitro incubation of naïve whole blood with trauma-hemorrhagic shock lymph replicated the in vivo trauma-hemorrhagic shock-induced RBC changes while 3) injection of trauma-hemorrhagic shock lymph into naïve animals recreated the RBC changes observed after actual trauma-hemorrhagic shock. CONCLUSIONS: 1) Trauma-hemorrhagic shock induces rapid RBC adhesion to endothelial cells in patients and animals. 2) Increased RBC CD36 expression characterizes the RBC-adhesive phenotype. 3) The RBC phenotypic and functional changes were induced by gut-derived humoral factors. These novel findings may explain the microvascular dysfunction occurring after trauma-hemorrhagic shock, sepsis, and other stress states.

Molecular pathology of pulmonary edema after injury in forensic autopsy cases.

The lung is vulnerable to trauma; pulmonary edema starts quickly as part of the systemic responses involved in shock. The present study investigated the molecular pathology of posttraumatic alveolar damage and responses involving pulmonary edema in forensic autopsy cases of injury (n = 66) compared with acute cardiac death cases (n = 13). Intrapulmonary mRNA and immunohistochemical expressions of matrix metalloproteinases (MMPs; MMP-2 and MMP-9), intercellular adhesion molecule-1, claudin-5, and aquaporins (AQPs, AQP-1 and AQP-5) were examined. Subacute injury deaths showed an increase in lung weight similar to that in acute cardiac death, but relative mRNA quantification using the Taqman real-time PCR assay demonstrated different findings among the causes of death; higher expressions were detected for all markers, except for AQP-5 in sharp instrument injury, for MMP-2 in blunt brain injury, and for MMP-9 in non-brain blunt injury, but these expression levels were lower in acute cardiac death. In immunostaining, only MMPs showed differences among the causes of death: MMP-2 expression was evident in most subacute deaths due to blunt brain injury and sharp instrument injury, whereas MMP-9 was intensely positive in those of non-brain blunt injury and sharp instrument injury. These findings suggest significant differences in the mechanism of pulmonary edema among fatal injuries and acute cardiac death, especially between blunt and sharp instrument injury. Systematic analysis of gene expressions using real-time PCR in combination with immunohistochemistry may be useful in evaluating pulmonary damage and responses after injury in death investigations, especially in connection with posttraumatic shock.

The Effects of Genetic 3-Mercaptopyruvate Sulfurtransferase Deficiency in Murine Traumatic-Hemorrhagic Shock.

INTRODUCTION: Hemorrhagic shock is a major cause of death after trauma. An additional blunt chest trauma independently contributes to mortality upon the development of an acute lung injury (ALI) by aggravating pathophysiological consequences of hemorrhagic shock. The maintenance of hydrogen sulfide availability is known to play an important role during hemorrhage and ALI. We therefore tested the impact of a genetic 3-mercaptopyruvate sulfurtransferase mutation (Δ3-MST) in a resuscitated murine model of traumatic-hemorrhagic shock. METHODS: Anesthetized wild-type (WT) and Δ3-MST mice underwent hemorrhagic shock with/without blunt chest trauma. Hemorrhagic shock was implemented for 1 h followed by retransfusion of shed blood and intensive care therapy for 4 h, including lung-protective mechanical ventilation, fluid resuscitation, and noradrenaline titrated to maintain a mean arterial pressure at least 50 mmHg. Systemic hemodynamics, metabolism, and acid-base status were assessed together with lung mechanics and gas exchange. Postmortem tissue samples were analyzed for immunohistological protein expression and mitochondrial oxygen consumption. RESULTS: 3-MST-deficient mice showed similar results in parameters of hemodynamics, gas exchange, metabolism, acid base status, and survival compared with the respective WT controls. Renal albumin extravasation was increased in Δ3-MST mice during hemorrhagic shock, together with a decrease of LEAK respiration in heart tissue. In contrast, mitochondrial oxygen consumption in the uncoupled state was increased in kidney and liver tissue of Δ3-MST mice subjected to the combined trauma. CONCLUSIONS: In summary, in a resuscitated murine model of traumatic-hemorrhagic shock, 3-MST deficiency had no physiologically relevant impact on hemodynamics and metabolism, which ultimately lead to unchanged mortality regardless of an additional blunt chest trauma.

Pulmonary tissue factor mRNA expression during murine traumatic shock: effect of P-selectin blockade.

Tissue factor (TF) is the primary cellular initiator of the coagulation protease cascade and serves as a cell surface receptor and a specific cofactor for plasma factors VII/VIIa. Because there is evidence that TF is regulated by a P-selectin dependent gene, we examined TF mRNA expression in the lungs during murine traumatic shock in the presence and absence of recombinant soluble P-selectin glycoprotein ligand-1 (rsPSGL.Ig) by using ribonuclease protection assays. Moreover, we studied the level of TF mRNA expression in mice with their P-selectin gene deleted (P-selectin -/-). Our data show that TF mRNA was significantly increased (+143%; P < 0.001) in the lungs 2 h after trauma compared with control rats subjected to sham trauma, which exhibited reduced TF mRNA expression (-34%; P < 0.001) after systemic administration of rsPSGL.Ig. The expression of TF mRNA was also significantly decreased (-29%; P < 0.05) in the lungs of P-selectin -/- mice compared with wild-type control C57B16 mice. The present results provide evidence for a P-selectin-dependent mechanism that enhances TF gene expression in traumatic shock. The major support for this mechanism is that either blockade of P-selectin by rsPSGL.Ig or deletion of the P-selectin gene leads to significant decreases in TF mRNA expression in the lung. These results are consistent with the concept that TF interacting with P-selectin may play a significant role in the pathophysiology of trauma.

Endothelin-1, endothelin receptors and ecNOS gene transcription in vital organs during traumatic shock in rats.

Endothelin-1 (ET-1) is a vasoconstrictor peptide that may play an important role in the pathophysiology of severe trauma. We examined ET-1 gene expression in vital organs (i.e., heart, lungs, kidneys, liver and small intestine) during murine traumatic shock using ribonuclease protection assays. Our data show that ET-1 mRNA was significantly increased in the lungs two hours after trauma when compared with control anesthetized rats. There was also a significant increase in ET-1 transcripts occurring in the kidneys, heart and liver. During these experimental conditions, we also observed statistically significant increased endothelin type B (ET(B)) receptor mRNA expression in the lung, heart, liver, kidney and small intestine. Expression of endothelial constitutive nitric oxide synthase (ecNOS) gene, which is functionally coupled to ET(B) receptor, also was increased in vital organs during traumatic shock. Endothelin type A (ET(A)) receptor gene expression was slightly decreased in the lung, liver and small intestine. These results suggest that ET-1 and ET(B) mRNA expression are mainly increased in the lung and other vital organs and may play a functional role in the pathophysiology of murine traumatic shock.

[Effect of traumatic shock on the cytogenetic processes in the epithelial cells of the cornea and tongue and in the bone marrow cells of white rats]. / Vliianie tramaticheskogo shoka na tsitogeneticheskie protsessy v kletkakh épiteliia rogovitsy i iazyka i v kletkakh kostnogo mozga belykh krys.

Traumatic shock in male albino rats as induced by prolonged (4-hour) compression of soft tissues of the hind limbs. It was found that 3, 6 and 24 hours after the termination of compression the mitotic activity in the corneal epithelium decreased 9.8-, 4.7- and 3-fold, respectively. The level of pathological mitoses in experimental animals rose 2.4 - 3-fold. Experiments in colchicine demonstrated that inhibition of mitotic activity is not related to the changed rate of mitosis. In the tongue epithelium, the depression of cell division was revealed 3 and 6 hours after compression was terminated. The mitotic index fell 2.7- and 3.7-fold, respectively. Radioautography showed a significant decrease in DNA-synthetizing capacity of epithelial cells of the tongue 3 and 6 hours after the compression was terminated. Karyotypic analysis of bone marrow cells showed that chromosome aberrations increased 3.3-fold.

P-selectin is up-regulated in vital organs during murine traumatic shock.

Murine traumatic shock is associated with increased adherence of neutrophils to the vascular endothelium resulting in neutrophil infiltration and tissue damage. We examined the effects of trauma on the expression of the adhesion molecule P-selectin in several vital organs (i.e., heart, lungs, liver, kidneys, and small intestine) 2 h after induction of Noble-Collip drum trauma in anesthetized rats. Total RNA was extracted from these organs and P-selectin mRNA was quantified by RNase protection assays. P-selectin mRNA was significantly increased over control nontraumatized, anesthetized rats in all vital organs (P<0.05 or less), with the largest increase occurring in the lung (P<0.01). Immunohistochemical analysis showed increased expression of P-selectin protein in postcapillary venules of all vital organs after trauma. To further investigate the possible mechanisms of increased P-selectin mRNA transcription promoter activity during trauma, we quantified binding of proteins from nuclear extracts to the kappaB site (-218GGGGGTGACCC[-207]) of the P-selectin gene by electrophoretic mobility shift assay. We confirmed the results of NF-kappaB binding by demonstrating increases in p50 and p52, as well as decreases in IkappaB in cytoplasmic and nuclear extracts from the lungs of trauma rats by Western blotting. Increased activity of the transcription factor, nuclear factor kappaB (NF-kappaB), occurred in all vital organs of the trauma rats compared to sham-operated controls. Our findings suggest that severe trauma results in up-regulation of P-selectin at the transcriptional level, which is partly controlled by an NF-kappaB-responsive element in the region of the P-selectin promoter. This increased activation of NF-kappB binding may contribute to the widespread increases in P-selectin expression observed in several vital organs 2 h after trauma, which in turn may play a key role in the pathogenesis of traumatic shock.

Effect of recombinant soluble P-selectin glycoprotein ligand-1 on leukocyte-endothelium interaction in vivo. Role in rat traumatic shock.

-Traumatic shock induces profound pathophysiological alterations and initiates inflammatory reactions in many tissues, thus resulting in acute multiple organ damage (eg, intestine, pancreas, and liver). In the rat, Noble-Collip drum trauma increases P-selectin expression on the vascular endothelium as a result of loss of endothelium-derived NO. Here we postulated that blockade of the earliest steps in leukocyte adhesion (ie, leukocyte rolling) via administration of a recombinant soluble form of P-selectin glycoprotein ligand-1 (PSGL-1; the recombinant soluble form is rsPSGL.Ig) would attenuate selectin-mediated events observed in the rat during traumatic shock. Using intravital microscopy of the rat mesenteric microvasculature, we found that intravenous infusion of rsPSGL.Ig significantly inhibited leukocyte-endothelium interaction (ie, leukocyte rolling, adherence, and transmigration) induced by traumatic shock as well as by activation of the microvascular endothelium with 50 micromol/L NG-nitro-L-arginine methyl ester. Immunohistochemical detection of P-selectin on the mesenteric venular endothelial surface demonstrated that rsPSGL.Ig functionally neutralizes effects of P-selectin on the endothelial cell surface rather than attenuating P-selectin expression. Systemic administration of rsPSGL.Ig to traumatized rats prolonged survival time and survival rate, significantly attenuating ileal myeloperoxidase activity and significantly preserving mesenteric endothelial function. Furthermore, PSGL-1 mRNA levels were significantly increased in the blood of traumatized rats and were reduced after systemic administration of rsPSGL.Ig. Thus, soluble recombinant forms of PSGL-1 are able to ameliorate acute shock states by suppressing selectin-mediated leukocyte-endothelium interaction at both the functional and molecular levels.