Stanniocalcin-1 (STC1) is an endogenous glycoprotein whose anti-inflammatory effects occur through induction of uncoupling proteins to reduce oxidative stress. In this study, we tested the hypothesis that exogenous recombinant human STC1 (rhSTC1) protects against lipopolysaccharide (LPS)-induced acute lung injury in mice. Anesthetized C57BL/6 mice underwent intratracheal spraying of LPS (20 µg/10 g body wt), and lung injury was assessed 24h later by analyzing pulmonary edema, bronchoalveolar lavage fluid, and lung histopathology. Lung inflammation, oxidative stress, and expression of STC1 and its downstream uncoupling protein 2 (UCP2) were analyzed at specific time points. Expression of UCP2 was suppressed initially but was subsequently upregulated after STC1 elevation in response to intratracheal administration of LPS. Intratracheal rhSTC1 treatment 1h before or after LPS spraying significantly attenuated pulmonary inflammation, oxidative stress, cell apoptosis, and acute lung injury. Pretreatment with STC1 short interfering RNA 48 h before LPS spraying inhibited the expression of STC1 and UCP2 and significantly increased the extent of lung injury. These findings suggest that STC1 is an endogenous stress protein that may counteract LPS-induced lung injury by inhibiting the inflammatory cascade and inducing antioxidant and antiapoptotic mechanisms. However, the potential clinical application of STC1 and the direct linkage between UCP2 and LPS-induced lung injury remain to be further investigated.

Endoplasmic reticulum (ER) stress that disrupts ER function can occur in response to a wide variety of cellular stress factors leads to the accumulation of unfolded and misfolded proteins in the ER. Many studies have shown that ER stress amplified inflammatory reactions and was involved in various inflammatory diseases. However, little is known regarding the role of ER stress in hyperoxia-induced acute lung injury (HALI). This study investigated the influence of ER stress inhibitor, 4-phenyl butyric acid (4-PBA), in mice with HALI. Treatment with 4-PBA in the hyperoxia groups significantly prolonged the survival, decreased lung edema, and reduced the levels of inflammatory mediators, lactate dehydrogenase, and protein in bronchoalveolar lavage fluid, and increased claudin-4 protein expression in lung tissue. Moreover, 4-PBA reduced the ER stress-related protein expression, NF-κB activation, and apoptosis in the lung tissue. In in vitro study, 4-PBA also exerted a similar effect in hyperoxia-exposed mouse lung epithelial cells (MLE-12). However, when claudin-4 siRNA was administrated in mice and MLE-12 cells, the protective effect of 4-PBA was abrogated. These results suggested that 4-PBA protected against hyperoxia-induced ALI via enhancing claudin-4 expression.

A high fiber diet (HFD) and dietary supplementation with acetate have been reported to have beneficial effects in a variety of diseases. We investigated the effects of a HFD and acetate supplementation on the gut microbiota and hyperoxia-induced acute lung injury (HALI) in mice. Mice were fed a control diet, HFD, or acetate supplementation for three weeks, and their gut microbiome composition, lung tissues, and bronchoalveolar lavage fluid (BALF) were examined after exposure to ambient air or hyperoxia. Both the HFD and acetate supplementation modified the gut microbiota community and increased the proportion of acetate-producing bacteria in mice exposed to hyperoxia. The HFD and acetate supplementation also increased the abundance of Bacteroides acidifaciens and reduced gut dysbiosis according to the ratio of Firmicutes to Bacteroidetes. Compared with hyperoxia-exposed mice fed a control diet, both the HFD and acetate supplementation significantly increased the survival time while reducing the severity of pulmonary edema and the concentrations of protein and inflammatory mediators in BALF. Moreover, the HFD and acetate supplementation reduced the production of free radicals, attenuated NF-κB signaling activation, and decreased apoptosis in the lung tissues. Overall, this study indicates that a HFD or acetate supplementation reduces the severity of HALI through alterations in the gut microbiota to exert anti-inflammatory effects.

Background: Poloxamer 188 (P188) possesses anti-inflammatory properties and can help to maintain plasma membrane function. P188 has been reported to exert beneficial effects in the treatment of various disorders. However, the effects of P188 in ischemia/reperfusion (IR)-induced acute lung injury have not been examined. Methods: We investigated the ability of P188 to attenuate IR-induced acute lung injury in rats and hypoxia/reoxygenation (HR) injury in murine epithelial cells. Isolated perfused rat lungs were exposed to 40 min ischemia followed by 60 min reperfusion to induce IR injury. Results: IR led to lung edema, increased pulmonary arterial pressure, promoted lung tissue inflammation and oxidative stress, and upregulated the levels of TNF-α, IL-6 and CINC-1, and increased Lactic dehydrogenase (LDH) activity in bronchoalveolar lavage fluid. IR also downregulated the levels of inhibitor of κB (IκB-α), upregulated nuclear factor (NF)-κB (NF-κB), and promoted apoptosis in lung tissues. P188 significantly suppressed all these effects. In vitro, P188 also exerted a similar effect in murine lung epithelial cells exposed to HR. Furthermore, P188 reduced the number of propidium iodide-positive cells, maintained cell membrane integrity, and enhanced cell membrane repair following HR. Conclusion: We conclude that P188 protects against lung IR injury by suppressing multiple signaling pathways and maintaining cell membrane integrity.

Annexin A1 (AnxA1) is an endogenous protein that modulates anti-inflammatory processes, and its therapeutic potential has been reported in a range of inflammatory diseases. The effect of AnxA1 on ischemia-reperfusion (IR)-induced lung injury has not been examined. In this study, isolated, perfused rat lungs were subjected to IR lung injury induced by ischemia for 40 min, followed by reperfusion for 60 min. The rat lungs were randomly treated with vehicle (phosphate-buffered saline), and Ac2-26 (an active N-terminal peptide of AnxA1) with or without an N-formyl peptide receptor (FPR) antagonist N-Boc-Phe-Leu-Phe-Leu-Phe (Boc2). An in vitro study of the effects of Ac2-26 on human alveolar epithelial cells subjected to hypoxia-reoxygenation was also investigated. Administration of Ac2-26 in IR lung injury produced a significant attenuation of lung edema, pro-inflammatory cytokine production recovered in bronchoalveolar lavage fluid, oxidative stress, apoptosis, neutrophil infiltration, and lung tissue injury. Ac2-26 also decreased AnxA1 protein expression, inhibited the activation of nuclear factor-κB and mitogen-activated protein kinase pathways in the injured lung tissue. Finally, treatment with Boc2 abolished the protective action of Ac2-26. The results indicated that Ac2-26 had a protective effect against acute lung injury induced by IR, which may be via the activation of the FPR.

Systemic administration of perfluorocarbons (PFCs) reportedly attenuates acute lung injury induced by acid aspiration and phorbol myristate acetate. However, the effects of PFCs on ischemia-reperfusion (IR)-induced lung injury have not been investigated. Typical acute lung injury was induced in rats by 60 min of ischemia and 60 min of reperfusion in isolated and perfused rat lung model. Rat lungs were randomly assigned to receive PBS (control), 1 % FC-77, IR only, or IR with different doses of FC-77 (0.1 %, 0.5 %, or 1 %). Subsequently, bronchoalveolar lavage fluid (BALF), perfusate, and lung tissues were collected to evaluate the degree of lung injury. IR caused a significant increase in the following parameters: pulmonary arterial pressure, capillary filtration coefficient, lung weight gain, lung weight/body weight ratio, wet/dry lung weight ratio, and protein concentration in BALF. TNF-α and cytokine-induced neutrophil chemoattractant-1 concentrations in perfusate samples and MDA concentration and MPO activities in lung tissues were also significantly increased. Histopathology showed increased septal thickness and neutrophil infiltration in the lung tissues. Furthermore, NF-κB activity was significantly increased in the lungs. However, pretreatment with 1 % FC-77 prior to IR significantly attenuated the increases in these parameters. In conclusion, our results suggest that systemic FC-77 administration had a protective effect on IR-induced acute lung injury. These protective mechanisms may have been mediated by the inhibition of NF-κB activation and attenuation of subsequent inflammatory response.

Current treatments for ischemia-reperfusion (IR)-induced acute lung injury are limited. Mesenchymal stem cell-conditioned medium (CM) has been reported to attenuate lung injury. Neural crest stem cells (NCSCs), a type of multipotent stem cells, are more easily obtained than mesenchymal stem cells. We hypothesize that NCSC-CM has anti-inflammatory properties that could protect against IR-induced lung injury in rats. In this study, NCSC-CM was derived from rat NCSCs. Typical acute lung injury was induced by 30-min ischemia followed by 90-min reperfusion in adult male Sprague-Dawley rats. Bronchoalveolar lavage fluid (BALF) and lung tissues were collected to analyze the degree of lung injury after the experiment. NCSC-CM was administered before ischemia and after reperfusion. NCSC-CM treatment significantly attenuated IR-induced lung edema, as indicated by decreases in pulmonary vascular permeability, lung weight gain, wet to dry weight ratio, lung weight to body weight ratio, pulmonary arterial pressure, and protein level in BALF. The levels of tumor necrosis factor-α and interleukin-6 in the BALF were also significantly decreased. Additionally, NCSC-CM improved lung pathology and neutrophil infiltration in the lung tissue, and significantly suppressed nuclear factor (NF)-κB activity and IκB-α degradation in the lung. However, heating NCSC-CM eliminated these protective effects. Our experiment demonstrates that NCSC-CM treatment decreases IR-induced acute lung injury and that the protective mechanism may be attributable to the inhibition of NF-κB activation and the inflammatory response. Therefore, NCSC-CM may be a novel approach for treating IR-induced lung injury.

Various animal studies have shown beneficial effects of hypercapnia in lung injury. However, in patients with acute respiratory distress syndrome (ARDS), there is controversial information regarding the effect of hypercapnia on outcomes. The duration of carbon dioxide inhalation may be the key to the protective effect of hypercapnia. We investigated the effect of pre-treatment with inhaled carbon dioxide on lipopolysaccharide (LPS)-induced lung injury in mice. C57BL/6 mice were randomly divided into a control group or an LPS group. Each LPS group received intratracheal LPS (2 mg/kg); the LPS groups were exposed to hypercapnia (5% carbon dioxide) for 10 min or 60 min before LPS. Bronchoalveolar lavage fluid (BALF) and lung tissues were collected to evaluate the degree of lung injury. LPS significantly increased the ratio of lung weight to body weight; concentrations of BALF protein, tumor necrosis factor-α, and CXCL2; protein carbonyls; neutrophil infiltration; and lung injury score. LPS induced the degradation of the inhibitor of nuclear factor-κB-α (IκB-α) and nuclear translocation of NF-κB. LPS increased the surface protein expression of toll-like receptor 4 (TLR4). Pre-treatment with inhaled carbon dioxide for 10 min, but not for 60 min, inhibited LPS-induced pulmonary edema, inflammation, oxidative stress, lung injury, and TLR4 surface expression, and, accordingly, reduced NF-κB signaling. In summary, our data demonstrated that pre-treatment with 10-min carbon dioxide inhalation can ameliorate LPS-induced lung injury. The protective effect may be associated with down-regulation of the surface expression of TLR4 in the lungs.

Ischemia-reperfusion (IR)-induced acute lung injury (ALI) is implicated in several clinical conditions like lung transplantation, acute pulmonary embolism after thrombolytic therapy, re-expansion of collapsed lung from pneumothorax or pleural effusion, cardiopulmonary bypass and etc. Because mortality remains high despite advanced medical care, prevention and treatment are important clinical issues for IR-induced ALI. Vascular endothelial growth factor (VEGF) has a controversial role in ALI. We therefore conducted this study to determine the effects of anti-VEGF antibody in IR-induced ALI. In the current study, the IR-induced ALI was conducted in a rat model of isolated-perfused lung in situ in the chest. The animals were divided into the control, control + preconditioning anti-VEGF antibody (bevacizumab, 5mg/kg), IR, IR + preconditioning anti-VEGF antibody (1mg/kg), IR+ preconditioning anti-VEGF antibody (5mg/kg) and IR+ post-IR anti-VEGF antibody (5mg/kg) group. There were eight adult male Sprague-Dawley rats in each group. The IR caused significant pulmonary micro-vascular hyper-permeability, pulmonary edema, neutrophilic infiltration in lung tissues, increased tumor necrosis factor-α, and total protein concentrations in bronchoalveolar lavage fluid. VEGF and extracellular signal-regulated kinase (ERK) were increased in IR-induced ALI. Administration of preconditioning anti-VEGF antibody significantly suppressed the VEGF and ERK expressions and attenuated the IR-induced lung injury. This study demonstrates the important role of VEGF in early IR-induced ALI. The beneficial effects of preconditioning anti-VEGF antibody in IR-induced ALI include the attenuation of lung injury, pro-inflammatory cytokines, and neutrophilic infiltration into the lung tissues.

Background: Increasing evidence suggests that Fbxo3 signaling has an important impact on the pathophysiology of the inflammatory process. Fbxo3 protein inhibition has reduced cytokine-driven inflammation and improved disease severity in animal model of Pseudomonas-induced lung injury. However, it remains unclear whether inhibition of Fbxo3 protein provides protection in acute lung injury induced by ischemia-reperfusion (I/R). In this study, we investigated the protective effects of BC-1215 administration, a Fbxo3 inhibitor, on acute lung injury induced by I/R in rats. Methods: Lung I/R injury was induced by ischemia (40 min) followed by reperfusion (60 min). The rats were randomly assigned into one of six experimental groups (n = 6 rats/group): the control group, control + BC-1215 (Fbxo3 inhibitor, 0.5 mg/kg) group, I/R group, or I/R + BC-1215 (0.1, 0.25, 0.5 mg/kg) groups. The effects of BC-1215 on human alveolar epithelial cells subjected to hypoxia-reoxygenation (H/R) were also examined. Results: BC-1215 significantly attenuated I/R-induced lung edema, indicated by a reduced vascular filtration coefficient, wet/dry weight ratio, lung injury scores, and protein levels in bronchoalveolar lavage fluid (BALF). Oxidative stress and the level of inflammatory cytokines in BALF were also significantly reduced following administration of BC-1215. Additionally, BC-1215 mitigated I/R-stimulated apoptosis, NF-κB, and mitogen-activated protein kinase activation in the injured lung tissue. BC-1215 increased Fbxl2 protein expression and suppressed Fbxo3 and TNFR associated factor (TRAF)1-6 protein expression. BC-1215 also inhibited IL-8 production and NF-κB activation in vitro in experiments with alveolar epithelial cells exposed to H/R. Conclusions: Our findings demonstrated that Fbxo3 inhibition may represent a novel therapeutic approach for I/R-induced lung injury, with beneficial effects due to destabilizing TRAF proteins.

Ventilator-induced lung injury is associated with inflammatory mechanism and causes high mortality. The objective of this study was to discover the role of IL-33 and its ST2 receptor in acute lung injury induced by mechanical ventilator (ventilator-induced lung injury; VILI). Male Wistar rats were intubated after tracheostomy and received ventilation at 10 cm H2O of inspiratory pressure (PC10) by a G5 ventilator for 4 hours. The hemodynamic and respiratory parameters were collected and analyzed. The morphological changes of lung injury were also assessed by histological H&E stain. The dynamic changes of lung injury markers such as TNF-α and IL-1β were measured in serum, bronchoalveolar lavage fluid (BALF), and lung tissue homogenization by ELISA assay. During VILI, the IL-33 profile change was detected in BALF, peripheral serum, and lung tissue by ELISA analysis. The Il-33 and ST2 expression were analyzed by immunohistochemistry staining and western blot analysis. The consequence of VILI by H&E stain showed inducing lung congestion and increasing the expression of pro-inflammatory cytokines such as TNF-α and IL-1β in the lung tissue homogenization, serum, and BALF, respectively. In addition, rats with VILI also exhibited high expression of IL-33 in lung tissues. Interestingly, the data showed that ST2L (membrane form) was highly accumulated in the membrane fraction of lung tissue in the PC10 group, but the ST2L in cytosol was dramatically decreased in the PC10 group. Conversely, the sST2 (soluble form) was slightly decreased both in the membrane and cytosol fractions in the PC10 group compared to the control group. In conclusion, these results demonstrated that ST2L translocation from the cytosol to the cell membranes of lung tissue and the down-expression of sST2 in both fractions can function as new biomarkers of VILI. Moreover, IL-33/ST2 signaling activated by mechanically responsive lung injury may potentially serve as a new therapy target.

Hypercapnic acidosis (HCA) has protective effects in animal models of acute lung injury, but the mechanism underlying the effect of HCA is unclear. Heme oxygenase-1 (HO-1) is an antioxidant enzyme that protects tissue from inflammation injury. We investigated whether HO-1 contributes to the protective effects of HCA in ischemia-reperfusion (IR)-induced lung injury. Typical acute lung injury in rats was successfully induced by 40 min of ischemia and 90 min of reperfusion in an isolated perfused lung model. The rat lungs were randomly assigned to the control group, IR group or IR + HCA group with or without zinc protoporphyrin IX (ZnPP), an HO-1 activity inhibitor. At the end of the experiment, bronchoalveolar lavage fluid (BALF) and lung tissues were collected to evaluate the degree of lung injury. In in vitro experiments, HO-1 siRNA transfected A549 cells were exposed to a normoxic or hypoxia-reoxygenation (H/R) environment in the presence or absence of HCA. IR caused significant increases in the pulmonary arterial pressure, lung weight to body weight and wet/dry ratios, lung weight gain, capillary filtration coefficient, lung injury scores, neutrophil infiltration, and concentrations of protein and TNF-α in the BALF. IR also induced degradation of inhibitor of nuclear factor (NF)-κB-α, increased IκB kinase (IKK)-β phosphorylation and nuclear translocation of NF-κB, and up-regulated HO-1 expression and activity. Furthermore, IR decreased Bcl-2 protein expression and increased the number of active caspase-3 stained cells. HCA treatment enhanced HO-1 expression and activity, and accordingly reduced IKK-NF-κB signaling, inhibited apoptosis, and significantly attenuated IR-induced changes. Treatment with ZnPP partially blocked the protective effect of HCA. In addition, HO-1 siRNA significantly reversed HCA-mediated inhibition of NF-κB signaling in A549 cells subjected to H/R. In conclusion, the protective effect of HCA in IR lung injury in rats was mediated in part by the anti-inflammatory and anti-apoptotic action of HO-1.