Enteral administration of the protease inhibitor gabexate mesilate preserves vascular function in experimental trauma/hemorrhagic shock.

Preserving vascular function is crucial for preventing multiorgan failure and death in ischemic and low-pressure states such as trauma/hemorrhagic shock (T/HS). It has recently been reported that inhibiting circulating proteases released from the bowel to the circulation during T/HS may preserve vascular function and improve outcomes following T/HS. This study aimed to evaluate the role of the serine protease inhibitor gabexate mesilate (GM) in preserving vascular function during T/HS when given enterally. We studied the vascular reactivity of mesenteric arteries from male Wistar rats treated with enteral GM (10 mg/kg) (GM-treated, n = 6) or control (Shock-control, n = 6) following (T/HS) using pressure myography. Concentration-response curves of endothelial-dependent and endothelial-independent agonists (e.g., acetylcholine, sodium nitroprusside) ranging from 10-10 to 10-5 M were performed. In a second set of experiments, ex-vivo arteries from healthy rats were perfused with plasma from shocked animals from both groups and vascular performance was similarly measured. Arteries from the GM-treated group demonstrated a preserved concentration-response curve to the α1 adrenergic agonist phenylephrine compared to arteries from Shock-control animals (- logEC50: - 5.73 ± 0.25 vs. - 6.48 ± 0.2, Shock-control vs. GM-treated, p = 0.04). When perfused with plasma from GM-treated rats, healthy arteries exhibited an even greater constriction and sensitivity to phenylephrine (- logEC50: - 6.62 ± 0.21 vs. - 7.13 ± 0.21, Shock-control vs. GM-treated, p = 0.02). Enteral GM also preserved the endothelium-dependent vascular response to agonists following T/HS and limited syndecan-1 shedding as a marker of glycocalyx compromise (41.84 ± 9 vs. 17.63 ± 3.97 ng/mL, Shock-control vs. GM-treated, p = 0.02). Syndecan-1 cleavage was correlated with plasma trypsin-like activity (r2 = 0.9611). Enteral gabexate mesilate was able to maintain vascular function in experimental T/HS, which was reflected by improved hemodynamics (mean arterial pressure 50.39 ± 7.91 vs. 64.95 ± 3.43 mmHg, Shock-control vs. GM treated, p = 0.0001). Enteral serine protease inhibition may be a potential therapeutic intervention in the treatment of T/HS.

Enteral Tranexamic Acid Decreases Proteolytic Activity in the Heart in Acute Experimental Hemorrhagic Shock.

The mechanisms for cardiac injury after hemorrhagic shock (HS) are unresolved. We hypothesize that remote organ damage can be caused by uncontrolled pancreatic proteolytic activity, as enteral protease inhibition improves outcomes in experimental HS. Uncontrolled proteolysis in the heart may disrupt cardiac metabolism and adrenergic control with subsequent deleterious outcomes. To test this hypothesis, the heart rate-pressure product (RPP) as an index of myocardial oxygen consumption and the levels of fatty acid transporter proteins CD36 and FATP6 as surrogates for metabolic activity in the heart were measured in rats subjected to experimental HS (n = 6/group) with and without the enteral protease inhibitor tranexamic acid (TXA). Plasma troponin I and heart fatty acid-binding protein (HFABP) concentrations were measured as indices of myocardial damage. Expression of the adrenergic receptors ß1, α1D, and ß2 was also measured in the heart to determine the possible effects of shock with and without enteral TXA on the adrenergic control of heart function. Hemorrhagic shock was induced by reduction in mean arterial blood pressure to 35 mm Hg for 2 hours before reperfusion of shed blood. The RPP was maintained in shocked animals treated enterally with TXA but not in those subjected to HS alone; this group also demonstrated decreased HFABP and plasma troponin I levels. Serine protease (trypsin, chymotrypsin, and elastase) and matrix metalloproteinase (MMP)-2 and MMP-9 activity was elevated in cardiac tissue and plasma after HS and abrogated by enteral TXA. Levels of CD36, FATP6, ß1, α1D, and ß2 were also increased after HS in cardiac tissue, and the increases were mitigated by TXA treatment. These results suggest that increased proteolytic activity may contribute to cardiac injury after HS. Enteral TXA prevents these changes, indicating a potential therapeutic option in the management of shock with resultant cardiac injury.

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.

Heme Oxygenase-2 Localizes to Mitochondria and Regulates Hypoxic Responses in Hepatocytes.

Hypoxia occurs as a part of multiple disease states, including hemorrhagic shock. Adaptive responses occur within the cell to limit the consequences of hypoxia. This includes changes in mitochondrial respiration, stress-induced cell signaling, and gene expression that is regulated by hypoxia inducible factor-1α (HIF-1α). Heme oxygenase-2 (HO-2) has been shown to be involved in oxygen sensing in several cell types. The purpose of these experiments was to test the hypothesis that HO-2 is a critical regulator of mitochondrial oxygen consumption and reactive oxygen species (ROS) production to influence hypoxia-adaptive responses such as HIF-1α protein levels and JNK signaling. Methods and Results. In vitro studies were performed in primary mouse hepatocytes. HO-2, but not HO-1, was expressed in mitochondria at baseline. Decreased oxygen consumption and increased mitochondrial ROS production in response to hypoxia were dependent upon HO-2 expression. HO-2 expression regulated HIF-1α and JNK signaling in a mitochondrial ROS-dependent manner. Furthermore, knockdown of HO-2 led to increased organ damage, systemic inflammation, tissue hypoxia, and shock in a murine model of hemorrhage and resuscitation. Conclusion. HO-2 signaling plays a role in hypoxic signaling and hemorrhagic shock. This pathway may be able to be harnessed for therapeutic effects.

Metformin Exerts Beneficial Effects in Hemorrhagic Shock in An AMPKα1-Independent Manner.

Despite therapeutic advances in hemorrhagic shock, mortality from multiple organ failure remains high. AMP-activated protein kinase (AMPK) is involved in cellular energy homeostasis. Two catalytic subunits, α1 and α2, have been identified, with α1 subunit largely expressed in major organs. Here, we hypothesized that genetic deficiency of AMPKα1 worsens hemorrhage-induced multiple organ failure. We also investigated whether treatment with metformin, a clinically used drug for metabolic homeostasis, affords beneficial effects. AMPKα1 wild-type (WT) and knock-out mice (KO) were subjected to hemorrhagic shock by blood withdrawing followed by resuscitation with shed blood and Lactated Ringer's solution and treatment with vehicle or metformin. Mice were sacrificed at 3 h after resuscitation. Compared with vehicle-treated WT animals, KO animals exhibited a more severe hypotension, higher lung and liver injury and neutrophil infiltration, and higher levels of plasma inflammatory cytokines. Metformin treatment ameliorated organ injury and mean arterial blood pressure in both WT and KO mice, without affecting systemic cytokine levels. Furthermore, metformin treatment reduced liver lipid peroxidation and increased levels of complex II cosubstrate FAD and levels of ATP in WT and KO mice. Beneficial effects of metformin were associated with organ-specific nuclear-cytoplasmic shuttling and activation of liver kinase B1 and AMPKα2. Thus, our data suggest that AMPKα1 is an important regulator of hemodynamic stability and organ metabolic recovery during hemorrhagic shock. Our data also suggest that metformin affords beneficial effects, at least in part, independently of AMPKα1 and secondary to AMPKα2 activation, increase of Complex II function and reduction of oxidative stress.

Age-Dependent Changes in AMPK Metabolic Pathways in the Lung in a Mouse Model of Hemorrhagic Shock.

The development of multiple organ failure in patients with hemorrhagic shock is significantly influenced by patient age. Adenosine monophosphate-activated protein kinase (AMPK) is a crucial regulator of energy homeostasis, which coordinates metabolic repair during cellular stress. We investigated whether AMPK-regulated signaling pathways are age-dependent in hemorrhage-induced lung injury and whether AMPK activation by 5-amino-4-imidazole carboxamide riboside (AICAR) affords lung protective effects. Male C57/BL6 young mice (3-5 mo), mature adult mice (9-12 mo), and young AMPKα1 knockout mice (3-5 mo) were subjected to hemorrhagic shock by blood withdrawing, followed by resuscitation with shed blood and lactated Ringer's solution. Plasma proinflammatory cytokines were similarly elevated in C57/BL6 young and mature adult mice after hemorrhagic shock. However, mature adult mice exhibited more severe lung edema and neutrophil infiltration, and higher mitochondrial damage in alveolar epithelial type II cells, than did young mice. No change in autophagy was observed. At molecular analysis, the phosphorylation of the catalytic subunit AMPKα1 was associated with nuclear translocation of peroxisome proliferator-activated receptor γ co-activator-α in young, but not mature, adult mice. Treatment with AICAR ameliorated the disruption of lung architecture in mice of both ages; however, effects in mature adult mice were different than young mice and also involved inhibition of nuclear factor-κB. In young AMPKα1 knockout mice, AICAR failed to improve hypotension and lung neutrophil infiltration. Our data demonstrate that during hemorrhagic shock, AMPK-dependent metabolic repair mechanisms are important for mitigating lung injury. However, these mechanisms are less competent with age.

Inhibition of fatty acid synthase with C75 decreases organ injury after hemorrhagic shock.

BACKGROUND: Hemorrhagic shock is the primary cause of morbidity and mortality in the intensive care units in patients under the age of 35. Several organs, including the lungs, are seriously affected by hemorrhagic shock and inadequate resuscitation. Excess free fatty acids have shown to trigger inflammation in various disease conditions. C75 is a small compound that inhibits fatty acid synthase, a key enzyme in the control of fatty acid metabolism that also stimulates fatty acid oxidation. We hypothesized that C75 treatment would be protective against hemorrhagic shock. METHODS: Adult male Sprague-Dawley rats were cannulated with a femoral artery catheter and subjected to controlled bleeding. Blood was shed to maintain a mean arterial pressure of 30 mm Hg for 90 minutes, then resuscitated over 30 minutes with a crystalloid volume equal to twice the volume of shed blood. Fifteen minutes into the 30-minute resuscitation, the rats received either intravenous infusion of C75 (1 mg/kg body weight) or vehicle (20% dimethyl sulfoxide). Blood and tissue samples were collected 6 hours after resuscitation (ie, 7.5 hours after hemorrhage) for analysis. RESULTS: After hemorrhage and resuscitation, C75 treatment decreased the increase in serum free fatty acids by 48%, restored adenosine triphosphate levels, and stimulated carnitine palmitoyl transferase-1 activity. Administration of C75 decreased serum levels of markers of injury (aspartate aminotransferase, lactate, and lactate dehydrogenase) by 38%, 32%, and 78%, respectively. Serum creatinine and blood urea nitrogen were also decreased significantly by 38% and 40%, respectively. These changes correlated with decreases in neutrophil infiltration in the lung, evidenced by decreases in Gr-1-stained cells and myeloperoxidase activity and improved lung histology. Finally, administration of C75 decreased pulmonary mRNA levels of cyclooxygenase-2 and interleukin-6 by 87% and 65%, respectively. CONCLUSION: Administration of C75 after hemorrhage and resuscitation decreased the increase in serum free fatty acids, decreased markers of tissue injury, downregulated the expression of inflammatory mediators, and decreased neutrophil infiltration and lung injury. Thus, the dual action of inhibiting fatty acid synthesis and stimulating fatty acid oxidation by C75 could be developed as a promising adjuvant therapy strategy to protect against hemorrhagic shock.

Enhanced cyclooxygenase-2 activity leads to intestinal dysmotility following hemorrhagic shock

ABSTRACT PURPOSE: To test whether hemorrhagic shock (HS) increases the Cyclooxygenase-2 (COX-2) expression in the intestine and whether this enhanced COX-2 expression mediates the intestinal dysmotility after HS. METHODS: Male Wistar rats were randomly divided into HS sham group and HS group. At 180 min following HS establishment, the duodenum samples were harvested to assess the motility function, protein expression of COX-2 and the downstream products of COX-2, prostaglandins. RESULTS: Examination of motility function ex vivo showed that the contractile response to acetylcholine of smooth muscle strips of rats subjected to HS was significantly suppressed. A COX-2 inhibitor, NS-398, abolished this depressed contractile response after HS. Western blotting revealed an increased protein expression of COX-2 in intestinal tissues of HS rats. Immunohistochemical examination indicated that intestine tissues of HS rats were manifested by part of villous expansion and disruption, a large amount of COX-2 positive cells appearance in lamina propria and submucosa. Furthermore, the contents of prostaglandin E2 was significantly increased in intestinal tissues of HS rats. CONCLUSION: The enhanced COX-2/ prostaglandin E2 involves in the hemorrhagic shock induced intestinal dysmotility.

Inflammation and Edema in the Lung and Kidney of Hemorrhagic Shock Rats Are Alleviated by Biliary Tract External Drainage via the Heme Oxygenase-1 Pathway.

The lung and kidney are two organs that are easily affected by hemorrhagic shock (HS). We investigated roles of biliary tract external drainage (BTED) in inflammation and edema of the lung and kidney in HS and its relationship with the heme oxygenase-1 (HO-1) pathway. Rat models of HS were induced by drawing blood from the femoral artery until a mean arterial pressure (MAP) of 40 ± 5 mmHg was achieved. A MAP of 40 ± 5 mmHg was maintained for 60 min. Thirty-six Sprague-Dawley rats were randomized to the following groups: sham group; HS group; HS + zinc protoporphyrin IX (ZnPP), a specific HO-1 inhibitor, group; HS + BTED group; HS + BTED + ZnPP group; and HS + BTED + bile infusion (BI) group. HO-1 levels, aquaporin-1 levels, and ratios of dry/wet in the lung and kidney increased markedly after BTED, but tumor necrosis factor-α and myeloperoxidase levels in the lung and kidney decreased significantly after BTED under HS conditions. Under the condition that HO-1 was inhibited by ZnPP, all these effects induced by BTED disappeared in the lung and kidney. These results demonstrated that inflammation and edema of the lung and kidney of HS rats are alleviated by BTED via the HO-1 pathway.

Kupffer cell inactivation by carbon monoxide bound to red blood cells preserves hepatic cytochrome P450 via anti-oxidant and anti-inflammatory effects exerted through the HMGB1/TLR-4 pathway during resuscitation from hemorrhagic shock.

Red blood cell (RBC) transfusions for controlling hemorrhaging induce systemic ischemia reperfusion, resulting in a decrease in hepatic cytochrome P450 (CYP) levels. Carbon monoxide (CO), when bound to red blood cells (CO-RBC) has the potential to protect the hepatic CYP protein to produce a resuscitative effect in a hemorrhagic shock rat model. The aim of this study was to investigate the mechanism by which CO-RBC resuscitation from a massive hemorrhage protects against a decrease in hepatic CYP. In the early phase (∼1h) after a hemorrhage and RBC resuscitation, hepatic CYP protein levels were significantly decreased with increasing hepatic free heme levels, but were maintained by a pre-treatment of gadolinium chloride (GdCl3), a Kupffer cell inhibitor, and Trolox, an anti-oxidant agent, as well as CO-RBC resuscitation. Under these conditions, the production of reactive oxygen species (ROS) derived from activated Kupffer cells was increased, but this increase was suppressed by CO-RBC resuscitation. At a late phase (6∼24h), CYP mRNA levels decreased after hemorrhage and RBC resuscitation, but not in the case of CO-RBC resuscitation. The increases in plasma IL-6 and TNF-α levels were decreased by CO-RBC resuscitation via the suppression of the toll-like receptor-4 (TLR-4) and the expression of the high mobility group box-1 (HMGB-1). Hepatic CYP protection after a hemorrhage and CO-RBC resuscitation can be attributed to the inactivation of Kupffer cells, resulting in the suppression of ROS production in the early phase and the suppression of inflammatory cytokine production via the TLR-4/HMGB-1signal pathway in the late phase.

Biliary tract external drainage alleviates kidney injury in shock.

BACKGROUND: Kidney injury is common in hemorrhagic shock (HS). Kidney injury leads to a systemic increase in serum chemokines and cytokines and causes injuries to other vital organs. Our previous studies showed that vitamin C led to organ protection and inflammation inhibitory effects in rat models of HS via induction heme oxygenase-1 (HO-1). We also found that biliary tract external drainage (BTED) increased the expression levels of HO-1 in rat livers. We investigated roles of BTED in kidney injury and its relationship with the HO-1 pathway in HS in this research. METHODS: Rat models of HS were induced by drawing blood from the femoral artery. BTED was performed by inserting a catheter into the bile duct. Thirty-six Sprague-Dawley rats were randomized to sham group; HS group; zinc protoporphyrin IX (Znpp) group; BTED group; BTED + Znpp group, and BTED + bile infusion group. The expression levels of HO-1 in the kidney were analyzed by Western blotting. The expression levels of occludin messenger RNA in the kidney were analyzed by real-time reverse transcription-polymerase chain reaction. The expression levels of occludin in the kidney were analyzed by immunohistochemistry. Histology of renal was performed by hematoxylin and eosin staining. RESULTS: Occludin messenger RNA and protein levels in the kidney increased markedly after BTED under HS conditions. Renal histopathologic scores decreased significantly after BTED under HS conditions. Znpp significantly inhibited all mentioned effects. CONCLUSIONS: BTED alleviates kidney injury in rats of HS via the HO-1 pathway.

Enhanced cyclooxygenase-2 activity leads to intestinal dysmotility following hemorrhagic shock.

PURPOSE: To test whether hemorrhagic shock (HS) increases the Cyclooxygenase-2 (COX-2) expression in the intestine and whether this enhanced COX-2 expression mediates the intestinal dysmotility after HS. METHODS: Male Wistar rats were randomly divided into HS sham group and HS group. At 180 min following HS establishment, the duodenum samples were harvested to assess the motility function, protein expression of COX-2 and the downstream products of COX-2, prostaglandins. RESULTS: Examination of motility function ex vivo showed that the contractile response to acetylcholine of smooth muscle strips of rats subjected to HS was significantly suppressed. A COX-2 inhibitor, NS-398, abolished this depressed contractile response after HS. Western blotting revealed an increased protein expression of COX-2 in intestinal tissues of HS rats. Immunohistochemical examination indicated that intestine tissues of HS rats were manifested by part of villous expansion and disruption, a large amount of COX-2 positive cells appearance in lamina propria and submucosa. Furthermore, the contents of prostaglandin E2 was significantly increased in intestinal tissues of HS rats. CONCLUSION: The enhanced COX-2/ prostaglandin E2 involves in the hemorrhagic shock induced intestinal dysmotility.

Recombinant human brain natriuretic peptide attenuates trauma-/haemorrhagic shock-induced acute lung injury through inhibiting oxidative stress and the NF-κB-dependent inflammatory/MMP-9 pathway.

Acute lung injury (ALI) is one of the most serious complications in traumatic patients and is an important part of multiple organ dysfunction syndrome (MODS). Recombinant human brain natriuretic peptide (rhBNP) is a peptide with a wide range of biological activity. In this study, we investigated local changes in oxidative stress and the NF-κB-dependent matrix metalloproteinase-9 (MMP-9) pathway in rats with trauma/haemorrhagic shock (TH/S)-induced ALI and evaluated the effects of pretreatment with rhBNP. Forty-eight rats were randomly divided into four groups: sham operation group, model group, low-dosage rhBNP group and high-dosage rhBNP group (n = 12 for each group). Oxidative stress and MPO activity were measured by ELISA kits. MMP-9 activity was detected by zymography analysis. NF-κB activity was determined using Western blot assay. With rhBNP pretreatment, TH/S-induced protein leakage, increased MPO activity, lipid peroxidation and metalloproteinase (MMP)-9 activity were inhibited. Activation of antioxidative enzymes was reversed. The phosphorylation of NF-κB and the degradation of its inhibitor IκB were suppressed. The results suggested that the protection mechanism of rhBNP is possibly mediated through upregulation of anti-oxidative enzymes and inhibition of NF-κB activation. More studies are needed to further evaluate whether rhBNP is a suitable candidate as an effective inhaling drug to reduce the incidence of TH/S-induced ALI.

Carbon monoxide-bound red blood cell resuscitation ameliorates hepatic injury induced by massive hemorrhage and red blood cell resuscitation via hepatic cytochrome P450 protection in hemorrhagic shock rats.

Red blood cell (RBC) transfusions are the gold standard in cases of massive hemorrhage, but induce hepatic ischemia-reperfusion injury, a serious complication associated with hemorrhage and RBC resuscitation. Thus, the development of a novel resuscitable fluid that is not associated with hepatic ischemia-reperfusion injury would be desirable. It was reported that exogenous carbon monoxide (CO) treatment ameliorated hepatic ischemia-reperfusion injury accompanying liver transplantation. This suggests that transfusions with CO-bound RBC (CO-RBC) might protect against hepatic ischemia-reperfusion injury following massive hemorrhage and resuscitation compared with RBC resuscitation. To investigate this, we created a hemorrhagic shock model rat, followed by resuscitation with RBC and CO-RBC. Hepatic ischemia-reperfusion injury and the destruction of hepatic cytochrome P450 (CYP) were significantly ameliorated in the CO-RBC resuscitation group compared with the RBC resuscitation group. The free heme derived from the destruction of hepatic CYP was correlated with hepatic oxidation and injury, suggesting that CO-RBC was a major factor in the amelioration of hepatic ischemia-reperfusion injury induced by hemorrhage and resuscitation via hepatic CYP protection. These results indicate that CO-RBC has potential for use as a resuscitative fluid in blood transfusion and does not suffer from the limitations associated with the RBC transfusions that are currently in use.

Pharmacological preconditioning with vitamin C attenuates intestinal injury via the induction of heme oxygenase-1 after hemorrhagic shock in rats.

Pre-induction of heme oxygenase (HO)-1, which is regarded as an effective method of "organ preconditioning", exerts beneficial effects during hemorrhagic shock (HS). However, the available HO-1 inducers exhibit disadvantages such as toxicity or complex technical requirements. Therefore, a safe and convenient HO-1 inducer would be promising and could be exploited in the treatment of foreseeable hemorrhaging, such as prior to major surgery. Here we investigated the effect of vitamin C (VitC), a common antioxidant, on intestinal HO-1 expression and examined whether VitC pretreatment prevented HS related intestinal tissue injuries after HO-1 induction. First, we conducted an in vitro study and found that HO-1 expression in rat intestinal epithelial cells (IEC-6) was induced by non-toxic VitC in a time and concentration dependent manner, and the mechanism was related to the activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Next, we conducted an in vivo study and found that VitC induced intestinal HO-1 protein expression (mainly observed in the intestinal epithelial cells) and HO-1 activity in normal SD rats, and that these HO-1 levels were further enhanced by VitC in a rat model of HS. The HS related intestinal injuries, including histological damage, pro-inflammatory cytokine levels (tumor necrosis factor and interleukin-6), neutrophil infiltration and apoptosis decreased after VitC pretreatment, and this alleviating of organ injuries was abrogated after the inhibition of HO-1 activity by zinc protoporphyrin-IX. It was of note that VitC did little histological damage to the intestine of the sham rats. These data suggested that VitC might be applied as a safe inducer of intestinal HO-1 and that VitC pretreatment attenuated HS related intestinal injuries via the induction of HO-1.

Rho kinase acts as a downstream molecule to participate in protein kinase Cε regulation of vascular reactivity after hemorrhagic shock in rats.

Our previous study demonstrated that Rho kinase and protein kinase C (PKC) played important parts in the regulation of vascular reactivity after shock. Using superior mesenteric arteries (SMAs) from hemorrhagic shock rats and hypoxia-treated vascular smooth muscle cells (VSMCs), relationship of PKCε regulation of vascular reactivity to Rho kinase, as well as the signal transduction after shock, was investigated. The results showed that inhibition of Rho kinase with the Rho kinase-specific inhibitor Y-27632 antagonized the PKCε-specific agonist carbachol and highly expressed PKCε-induced increase of vascular reactivity in SMAs and VSMCs, whereas inhibition of PKCε with its specific inhibitory peptide did not antagonize the Rho kinase agonist (U-46619)-induced increase of vascular reactivity in SMAs and VSMCs. Activation of PKCε or highly expressed PKCε upregulated the activity of Rho kinase and the phosphorylation of PKC-dependent phosphatase inhibitor 17 (CPI-17), zipper interacting protein kinase (ZIPK), and integrin-linked kinase (ILK), whereas activation of Rho kinase increased only CPI-17 phosphorylation. The specific neutralization antibodies of ZIPK and ILK antagonized PKCε-induced increases in the activity of Rho kinase, but CPI-17 neutralization antibody did not antagonize this effect. These results suggested that Rho kinase takes part in the regulation of PKCε on vascular reactivity after shock. Rho kinase is downstream of PKCε. Protein kinase Cε activates Rho kinase via ZIPK and ILK; CPI-17 is downstream of Rho kinase.

Mesenteric lymph diversion abrogates 5-lipoxygenase activation in the kidney following trauma and hemorrhagic shock.

BACKGROUND: Early acute kidney injury (AKI) following trauma is associated with multiorgan failure and mortality. Leukotrienes have been implicated both in AKI and in acute lung injury. Activated 5-lipoxygenase (5-LO) colocalizes with 5-LO-activating protein (FLAP) in the first step of leukotriene production following trauma and hemorrhagic shock (T/HS). Diversion of postshock mesenteric lymph, which is rich in the 5-LO substrate of arachidonate, attenuates lung injury and decreases 5-LO/FLAP associations in the lung after T/HS. We hypothesized that mesenteric lymph diversion (MLD) will also attenuate postshock 5-LO-mediated AKI. METHODS: Rats underwent T/HS (laparotomy, hemorrhagic shock to a mean arterial pressure of 30 mm Hg for 45 minutes, and resuscitation), and MLD was accomplished via cannulation of the mesenteric duct. Extent of kidney injury was determined via histology score and verified by urinary neutrophil gelatinase-associated lipocalin assay. Kidney sections were immunostained for 5-LO and FLAP, and colocalization was determined by fluorescence resonance energy transfer signal intensity. The end leukotriene products of 5-LO were determined in urine. RESULTS: AKI was evident in the T/HS group by derangement in kidney tubule architecture and confirmed by neutrophil gelatinase-associated lipocalin assay, whereas MLD during T/HS preserved renal tubule morphology at a sham level. MLD during T/HS decreased the associations between 5-LO and FLAP demonstrated by fluorescence resonance energy transfer microscopy and decreased leukotriene production in urine. CONCLUSION: 5-LO and FLAP colocalize in the interstitium of the renal medulla following T/HS. MLD attenuates this phenomenon, which coincides with pathologic changes seen in tubules during kidney injury and biochemical evidence of AKI. These data suggest that gut-derived leukotriene substrate predisposes the kidney and the lung to subsequent injury.

Bkca opener, NS1619 pretreatment protects against shock-induced vascular hyporeactivity through PDZ-Rho GEF-RhoA-Rho kinase pathway in rats.

BACKGROUND: Our previous study showed that the ischemic preconditioning and pretreatment of adenosine triphosphate-sensitive potassium channel (KATP) opener, pinacidil, may induce a good protective effect on shock-induced vascular hyporeactivity. Whether the pretreatment of opener/activator of the large-conductance calcium-activated potassium channel (Bkca), NS1619, can also induce a protective effect on vascular reactivity and play a beneficial effect on subsequent hemorrhagic shock is not clear. METHODS: With Sprague-Dawley rats subjected to hemorrhagic shock and their isolated superior mesenteric artery, the protective effect of NS1619 (0.5, 1, 2, and 4 mg/kg) pretreatment (30 minutes before hemorrhage shock) on vascular reactivity and the underlying mechanisms were observed. RESULTS: NS1619 pretreatment significantly improved the 72-hour survival of hemorrhagic shock rats, alleviated shock-induced decrease of vascular reactivity and calcium sensitivity, and increased the cardiac output and oxygen delivery. NS1619 2 mg/kg had the best effect. These protective effects of NS1619 pretreatment on vascular reactivity and calcium sensitivity were antagonized by RhoA inhibitor, C3 transferase, and Rho kinase antagonist, Y-27632. NS1619 pretreatment up-regulated the activities of RhoA, Rho-kinase, and PDZ-Rho GEF (guanine nucleotide exchange factor). These effects of NS1619 pretreatment were eliminated by RhoA inhibitor, C3 transferase. CONCLUSION: Bkca opener, NS1619 pretreatment has good protective effect on vascular reactivity and calcium sensitivity, which plays a good beneficial effect on hemorrhagic shock. The mechanism may be mainly through PDZ-Rho GEF-RhoA-Rho kinase pathway. Bkca channel may be a potential target for the treatment of shock-induced vascular hyporeactivity.

Pancreatic digestive enzyme blockade in the small intestine prevents insulin resistance in hemorrhagic shock.

Hemorrhagic shock is associated with metabolic defects, including hyperglycemia and insulin resistance, but the mechanisms are unknown. We recently demonstrated that reduction of the extracellular domain of the insulin receptor by degrading proteases may lead to a reduced ability to maintain normal plasma glucose values. In shock, transfer of digestive enzymes from the lumen of the intestine into the systemic circulation after breakdown of the intestinal mucosal barrier causes inflammation and organ dysfunction. Suppression of the digestive enzymes in the lumen of the intestine with protease inhibitors is effective in reducing the level of the inflammatory reactions. To determine the degree to which blockade of digestive enzymes affects insulin resistance in shock, rats were exposed to acute hemorrhagic shock (mean arterial pressure of 30 mmHg for 2 h) at which time all shed blood volume was returned. Digestive proteases in the intestine were blocked with a serine protease inhibitor (tranexamic acid in polyethylene glycol and physiological electrolyte solution), and the density of the insulin receptor was measured with immunohistochemistry in the mesentery microcirculation. The untreated rat without enzyme blockade had significantly attenuated levels of insulin receptor density as compared with control and treated rats. Blockade of the digestive proteases after 60 min of hypotension in the lumen of the small intestine led to a lesser decrease in insulin receptor density compared with controls without protease blockade. Glucose tolerance test indicates a significant increase in plasma glucose levels 2 h after hemorrhagic shock, which are reduced to control values in the presence of protease inhibition in the lumen of the intestine. The transient reduction of the plasma glucose levels after an insulin bolus is significantly attenuated after shock but is restored when digestive enzymes in the lumen of the intestine are blocked. These results suggest that in hemorrhagic shock elevated microvascular extracellular digestive enzyme activity causes insulin receptor dysfunction, hyperglycemia, and reduced ability to regulate blood glucose values.