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Multiorgan failure in hemorrhagic shock is triggered by gut barrier dysfunction and consequent systemic infiltration of proinflammatory factors. Our previous study has shown that diphenyldihaloketone drugs 4-[3,5-bis[(2-chlorophenyl)methylene]-4-oxo-1-piperidinyl]-4-oxo-2-butenoic acid (CLEFMA) and 3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone (EF24) restore gut barrier dysfunction and reduce systemic inflammatory response in hemorrhagic shock. We investigated the effect of hemorrhagic shock on proteasome activity of intestinal epithelium and how CLEFMA and EF24 treatments modulate proteasome function in hemorrhagic shock. CLEFMA or EF24 (0.4 mg/kg) were given 1 h after withdrawing 50% of blood from Sprague-Dawley rats; no other resuscitation was provided. After another 5 h of compensation, small gut was collected to process tissue for proteasome activity, immunoblotting, and mRNA levels of genes responsible for unfolded-protein response (XBP1, ATF4, glucose-regulated protein of 78/95 kDa, and growth arrest and DNA damage inducible genes 153/34), polyubiquitin B and C, and immunoproteasome subunits β type-8 and -10 and proteasome activator subunit 1. We found that hemorrhagic shock induced proteasome activity in gut tissue and reduced the amounts of ubiquitinated proteins displayed on antiubiquitin immunoblots. However, simultaneous induction of unfolded-protein response or immunoproteasome genes was not observed. CLEFMA and EF24 treatments abolished the hemorrhagic shock-induced increase in proteasome activity. Further investigations revealed that the induction of proteasome in hemorrhagic shock is associated with disassembly of 26S proteasome; CLEFMA and EF24 prevented this disassembly. Consistent with these data, CLEFMA and EF24 reduced hemorrhagic shock-induced degradation of 20S substrate ornithine decarboxylase in gut tissue. These results suggest that activated proteasome plays an important role in ischemic gut pathophysiology, and it can be a druggable target in shock-induced gut dysfunction. NEW & NOTEWORTHY Ischemic injury to the gut is a trigger for the systemic inflammatory response and multiple organ failure in trauma and hemorrhagic shock. We show for the first time that hemorrhagic shock induces the gut proteasome activity by engendering 26S proteasome disassembly. Diphenyldihaloketones 4-[3,5-bis[(2-chlorophenyl)methylene]-4-oxo-1-piperidinyl]-4-oxo-2-butenoic acid and 3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone treatment prevented the 26S disassembly. Understanding the role of proteasome in shock-associated gut injury will assist in the development of therapeutic means to address it.

Gut barrier dysfunction is the major trigger for multiorgan failure associated with hemorrhagic shock (HS). Although the molecular mediators responsible for this dysfunction are unclear, oxidative stress-induced disruption of proteostasis contributes to the gut pathology in HS. The objective of this study was to investigate whether resuscitation with nanoparticulate liposome-encapsulated hemoglobin (LEH) is able to restore the gut proteostatic mechanisms. Sprague-Dawley rats were recruited in four groups: control, HS, HS+LEH, and HS+saline. HS was induced by withdrawing 45% blood, and isovolemic LEH or saline was administered after 15 min of shock. The rats were euthanized at 6 h to collect plasma and ileum for measurement of the markers of oxidative stress, unfolded protein response (UPR), proteasome function, and autophagy. HS significantly increased the protein and lipid oxidation, trypsin-like proteasome activity, and plasma levels of IFNγ. These effects were prevented by LEH resuscitation. However, saline was not able to reduce protein oxidation and plasma IFNγ in hemorrhaged rats. Saline resuscitation also suppressed the markers of UPR and autophagy below the basal levels; the HS or LEH groups showed no effect on the UPR and autophagy. Histological analysis showed that LEH resuscitation significantly increased the villus height and thickness of the submucosal and muscularis layers compared with the HS and saline groups. Overall, the results showed that LEH resuscitation was effective in normalizing the indicators of proteostasis stress in ileal tissue. On the other hand, saline-resuscitated animals showed a decoupling of oxidative stress and cellular protective mechanisms., (Copyright © 2016 the American Physiological Society.)

Maternal hypothyroidism affects postnatal lung structure. High prevalence of hypothyroxinemia (low T4, normal T3) in iodine-deficient pregnant women and associated risk for neuropsychological development along with high infant/neonatal mortality ascribed to respiratory distress prompted us to study the effects of maternal hypothyroxinemia on postnatal lung development. Female Sprague Dawley rats were given a low-iodine diet (LID) with 1% KClO(4) in drinking water for 10 days, to minimize thyroid hormone differences. Half of these rats were continued on iodine-deficient diet; ID (LID with 0.005% KClO(4)) for 3 mo, whereas the rest were switched to an iodine-sufficient diet; IS [LID + potassium iodide (10 μg iodine/20 g of diet + normal drinking water)]. Pups born to ID mothers were compared with age-matched pups from IS mothers at postnatal days 8 (P8) and 16 (P16) (n = 6-8/group). ID pups had normal circulating T3 but significantly low T4 levels (P < 0.05) and concomitantly approximately sixfold higher thyroid hormone receptor-β mRNA in alveolar epithelium. Lung histology revealed larger and irregularly shaped alveoli in ID pups relative to controls. Lung function was assessed at P16 using a double-chambered plethysmograph and observed reduced tidal volume, peak inspiratory and expiratory flow, and dynamic lung compliance in ID pups compared with IS pups. Significant lowering of surfactant protein (SP)-B and SP-C mRNA and protein found in ID pups at P16. ID pups had 16-fold lower matrix metalloproteinase-9 mRNA levels in their alveolar epithelium. In addition, mRNA levels of thyroid transcription factor-1 and SP-D were significantly higher (3-fold) compared with IS pups. At P16, significantly lower levels of SP-B and SP-C found in ID pups may be responsible for immature lung development and reduced lung compliance. Our data suggest that maternal hypothyroxinemia may result in the development of immature lungs that, through respiratory distress, could contribute to the observed high infant mortality in ID neonates.