Adenosine diphosphate ribosylation factor 6 inhibition protects burn sepsis induced lung injury through preserving vascular integrity and suppressing ASC inflammasome transmission.

Severe burns are devastating injuries with significant immune dysfunction and result in substantial mortality and morbidity due to sepsis induced organ failure. Acute lung injury is the most common type of organ injury in sepsis, however, the mechanisms of which are poorly understood and effective therapeutic measures are limited. This study is aimed to investigate the effect of a small Guanosine triphosphatase (GTPase), Adenosine diphosphate ribosylation factor 6 (ARF6), on burn sepsis induced lung injury, and discuss the possible mechanisms. Burn sepsis was established in male C57BL/6 mice. Mice were anesthetised by intramuscular injection of ketamine and xylazine hydrochloride, then 30% TBSA full thickness burn followed by sub-eschar injection of lipopolysaccharide. Animals were treated with intraperitoneal injection of a small molecule inhibitor of ARF6: NAV-2729, or vehicle, right after the burn and sepsis stimuli were inflicted. Lung tissues were harvested for histopathological observation and the acute lung injury scores were calculated. Organ permeability, Vascular Endothelial Cadherin (VE-cadherin) expression, inflammatory cytokine levels and myeloperoxidase activity in lung tissues were detected. Rat pulmonary microvascular endothelial cells (PMVECs) were stimulated by burn sepsis serum with or without 10 μM NAV-2729. The ARF6 activation, VE-cadherin expression, inflammasome activity, adapter protein apoptosis speck-like protein containing a caspase recruiting domain (ASC) specks and cytokines secretion were determined. Student's t test was used for comparison between two groups. Multiple comparisons among groups were performed by using analysis of variance, with Tukey's test for the post hoc test. NAV-2729 treatment attenuated burn sepsis induced lung injury and promoted survival of burn septic mice by preserving VE-cadherin expression in endothelial cell adherent junction and limited vascular hyperpermeability in lung tissues. Moreover, inflammatory cytokine expression and inflammatory injury in lung tissues were alleviated. Mechanistically, NAV-2729 enhanced vascular integrity by inhibiting ARF6 activation and restoring VE-cadherin expression in PMVECs. In addition, NAV-2729 inhibited ARF6-dependent phagocytosis of ASC specks, thus preventing inflammation propagation mediated by cell-to-cell transmission of ASC specks. ARF6 inhibition preserved vascular integrity by restoring expression of VE-cadherin and suppressed the spread of inflammation by affecting phagocytosis of ASC specks, thus protected against sepsis induced lung injury and improve survival of burn septic animals. The findings of this study implied potential therapeutics by which ARF6 inhibition can protect lung function from septic induced lung injury and improve outcomes in burn sepsis.

Xiao M ，Zhang P ，Chen Z ，Liu X ，Wei W ，He Z ，Wang Y ，Cheng J ，Zhu Z ，Wen J ，Yang H ... -  《-》

Sirt3 Maintains Microvascular Endothelial Adherens Junction Integrity to Alleviate Sepsis-Induced Lung Inflammation by Modulating the Interaction of VE-Cadherin and β-Catenin.

Inflammatory injury is a hallmark of sepsis-induced acute respiratory distress syndrome (ARDS)/acute lung injury (ALI). However, the mechanisms underlying inflammatory injury remain obscure. Here, we developed the novel strategy to suppress lung inflammation through maintaining microvascular endothelial barrier integrity. VE-cadherin is the main adherens junction protein that interacts with β-catenin and forms a complex. We found that lung inflammation was accompanied by decreased VE-cadherin expression and increased β-catenin activity in animal models and human pulmonary microvascular endothelial cells (HPMECs), illuminating the relationship among VE-cadherin/β-catenin complex, microvascular endothelial barrier integrity, and inflammation. Furthermore, we showed that the VE-cadherin/β-catenin complex dissociated upon lung inflammation, while Sirt3 promoted the stability of such a complex. Sirt3 was decreased during lung inflammation in vivo and in vitro. Sirt3 deficiency not only led to the downregulation of VE-cadherin but also enhanced the transcriptional activity of β-catenin that further increased β-catenin target gene MMP-7 expression, thereby promoting inflammatory factor COX-2 expression. Sirt3 overexpression promoted VE-cadherin expression, inhibited β-catenin transcriptional activity, strengthened the stability of the VE-cadherin/β-catenin complex, and suppressed inflammation in HPMECs. Notably, Sirt3 deficiency significantly damaged microvascular endothelial barrier integrity and intensified lung inflammation in animal model. These results demonstrated the role of Sirt3 in modulating microvascular endothelial barrier integrity to inhibit inflammation. Therefore, strategies that aim at enhancing the stability of endothelial VE-cadherin/β-catenin complex are potentially beneficial for preventing sepsis-induced lung inflammation.

Chen DQ ，Shen MJ ，Wang H ，Li Y ，Tang AL ，Li S ，Xiong MC ，Guo Y ，Zhang GQ ... -  《-》

Inhibition of MRP4 alleviates sepsis-induced acute lung injury in rats.

This study was undertaken to examine the regulatory role of multidrug resistance-associated protein 4 (MRP4) in an experimental model of sepsis-induced acute lung injury in rats. Sepsis was induced by cecal ligation and puncture in anesthetized rats. Animals were then randomly assigned to receive intravenous injection of vehicle or MRP4 inhibitor (MK571, 20 mg/kg). The pathological changes were observed by hematoxylin and eosin staining. Lung water content, lung vascular permeability and inflammatory cell count in bronchoalveolar lavage fluid (BALF) were quantified. Serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were measured. In addition, lung tissue cyclic adenosine monophosphate (cAMP) levels were examined by enzyme-linked immunosorbent assay. Furthermore, the effects of MRP4 knockdown on lipopolysaccharide (LPS)-induced endothelial permeability and the cytoskeleton of rat pulmonary microvascular endothelial cells (PMVECs) were detected. The protein expression levels of MRP4, Rac1, VE-cadherin, β-catenin and ZO-1 were measured by Western blot analysis. MK571 significantly reduced lung tissue damage, lung water content and lung vascular permeability. Lung tissue cAMP levels were attenuated in MK571-treated animals compared with vehicle controls. MK571 also decreased sepsis-induced inflammatory cell accumulation in BALF. In addition, the MK571 group had significantly lower serum TNF-α and IL-6 levels compared with vehicle controls. Consistently, knockdown of MRP4 protected against LPS-induced increase in the endothelial permeability and the destruction of cytoskeleton in vitro. Furthermore, silencing MRP4 gene significantly reduced MRP4 protein expression and restored the protein expression of Rac1, VE-cadherin, β-catenin and ZO-1 in rat PMVECs in response to LPS stimulation. These data suggest that inhibition of MRP4 significantly alleviates sepsis-induced acute lung injury in rats.

Xia W ，Zhang H ，Pan Z ，Li G ，Zhou Q ，Hu D ，Liu Y ... -  《-》

HYDROGEN PREVENTS LIPOPOLYSACCHARIDE-INDUCED PULMONARY MICROVASCULAR ENDOTHELIAL CELL INJURY BY INHIBITING STORE-OPERATED Ca 2+ ENTRY REGULATED BY STIM1/ORAI1.

Background: Sepsis is a type of life-threatening organ dysfunction that is caused by a dysregulated host response to infection. The lung is the most vulnerable target organ under septic conditions. Pulmonary microvascular endothelial cells (PMVECs) play a critical role in acute lung injury (ALI) caused by severe sepsis. The impairment of PMVECs during sepsis is a complex regulatory process involving multiple mechanisms, in which the imbalance of calcium (Ca 2+ ) homeostasis of endothelial cells is a key factor in its functional impairment. Our preliminary results indicated that hydrogen gas (H 2 ) treatment significantly alleviates lung injury in sepsis, protects PMVECs from hyperpermeability, and decreases the expression of plasma membrane stromal interaction molecule 1 (STIM1), but the underlying mechanism by which H 2 maintains Ca 2+ homeostasis in endothelial cells in septic models remains unclear. Thus, the purpose of the present study was to investigate the molecular mechanism of STIM1 and Ca 2+ release-activated Ca 2+ channel protein1 (Orai1) regulation by H 2 treatment and explore the effect of H 2 treatment on Ca 2+ homeostasis in lipopolysaccharide (LPS)-induced PMVECs and LPS-challenged mice. Methods: We observed the role of H 2 on LPS-induced ALI of mice in vivo . The lung wet/dry weight ratio, total protein in the bronchoalveolar lavage fluid, and Evans blue dye assay were used to evaluate the pulmonary endothelial barrier damage of LPS-challenged mice. The expression of STIM1 and Orai1 was also detected using epifluorescence microscopy. Moreover, we also investigated the role of H 2 -rich medium in regulating PMVECs under LPS treatment, which induced injury similar to sepsis in vitro . The expression of STIM1 and Orai1 as well as the Ca 2+ concentration in PMVECs was examined. Results:In vivo , we found that H 2 alleviated ALI of mice through decreasing lung wet/dry weight ratio, total protein in the bronchoalveolar lavage fluid and permeability of lung. In addition, H 2 also decreased the expression of STIM1 and Orai1 in pulmonary microvascular endothelium. In vitro , LPS treatment increased the expression levels of STIM1 and Orai1 in PMVECs, while H 2 reversed these changes. Furthermore, H 2 ameliorated Ca 2+ influx under sepsis-mimicking conditions. Treatment with the sarco/endoplasmic reticulum Ca 2+ adenosine triphosphatase inhibitor, thapsigargin, resulted in a significant reduction in cell viability as well as a reduction in the expression of junctional proteins, including vascular endothelial-cadherin and occludin. Treatment with the store-operated Ca 2+ entry inhibitor, YM-58483 (BTP2), increased the cell viability and expression of junctional proteins. Conclusions: The present study suggested that H 2 treatment alleviates LPS-induced PMVEC dysfunction by inhibiting store-operated Ca 2+ entry mediated by STIM1 and Orai1 in vitro and in vivo .

Li Y ，Chen H ，Shu R ，Zhang X ，Wang G ，Yin Y ... -  《-》

[Mechanism of Extracellular Histone-Induced Endothelial Dysfunction Leading to Sepsis-Induced Acute Respiratory Distress Syndrome].

Sepsis-induced acute respiratory distress syndrome (ARDS) is an independent risk factor for mortality in critically ill septic patients. However, effective therapeutic targets are still unavailable due to the lack of understanding of its unclear pathogenesis. With increasing understanding in the roles of circulating histones and endothelial dysfunction in sepsis, we aimed to investigate the mechanism of histone-induced endothelial dysfunction leading to sepsis-induced ARDS and to provide experimental support for histone-targeted treatment of sepsis-induced ARDS. First of all, in vitro experiments were conducted. Human umbilical vein endothelial cells (HUVEC) were stimulated with gradient concentrations of histones to explore for the optimal stimulation concentration in vitro. Then, HUVEC were exposed to histones at an optimal concentration with or without resatorvid (TAK-242), a selective inhibitor of Toll-like receptor 4 (TLR4), for 24 hours for modeling. The cells were divided into 4 groups: 1) the blank control group, 2) the blank control+TAK-242 intervention group, 3) the histone stimulation group, and 4) the histone+TAK-242 intervention group. HUVEC apoptosis was determined by flow cytometry, VE-Cadherin expression in endothelial cells was determined by Western blot, and the integrity of adhesion connections between endothelial cells was evaluated with confocal fluorescence microscopic images. Male C57BL/6 mice aged 6-8 weeks and weighing 22-25 g were used for the in vivo experiment. Then, the mice were given cecal ligation and puncture (CLP) as well as histone injection at 50 mg/kg via the tail vein for sepsis modeling. The experimental animals were divided into 6 groups: 1) the blank control group, 2) the blank control+TAK-242 intervention group, 3) the CLP model group, 4) the CLP+TAK-242 intervention group, 5) the histone model group, and 6) the histone+TAK-242 intervention group. After 24 h, the concentrations of serum interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined using ELISA kits. Western blot was performed to determine the expression of vascular endothelial (VE)-cadherin in the lung tissue. Hematoxylin and eosin (HE) staining was performed to observe the pathological changes in the lung tissue of the mice. Evans Blue was injected via the tail vein 30 min before the mice were sacrificed. Lung tissue was collected after the mice were sacrificed. Then, the concentrations of Evans blue dye per unit mass in the lung tissue from mice of different groups were evaluated, the rates of pulmonary endothelial leakage were calculated, and the integrity of the pulmonary endothelial barrier was evaluated. The results of the in vitro experiment showed that, compared with those of the control group, HUVEC apoptosis was significantly increased under histone stimulation (P<0.05), the expression of VE-cadherin was decreased (P<0.05), and the integrity of adherens junctions between endothelial cells was damaged. TAK-242 can significantly inhibit histone-induced HUVEC apoptosis and VE-cadherin expression reduction and maintain the integrity of adherens junctions between endothelial cells. According to the findings from the in vivo experiments, in mice with CLP-induced and histone-induced sepsis, TAK-242 effectively alleviated the increase in serum concentrations of IL-6 and TNF-α, reduced the downregulation of VE-cadherin expression in the lung tissue (P<0.05), decreased endothelial permeability of the lung vessels, and improved pathological injury in the lung tissue. By binding to TLR-4, histone decreases VE-cadherin expression on the surface of vascular endothelial cells, disrupts the integrity of intercellular adherens junctions, and triggers pathological damage to lung tissue. Using TLR-4 inhibitors can prevent sepsis-induced ARDS in histone-induced sepsis.

Yang T ，Li Y ，Su B 《-》