Adrenomedullin expression in a rat model of acute lung injury induced by hypoxia and LPS

Adrenomedullin (ADM) is upregulated independently by hypoxia and LPS, two key factors in the pathogenesis of acute lung injury (ALI). This study evaluates the expression of ADM in ALI using experimental models combining both stimuli: an in vivo model of rats treated with LPS and acute normobaric hypoxia (9% O2) and an in vitro model of rat lung cell lines cultured with LPS and exposed to hypoxia (1% O2). ADM expression was analyzed by in situ hybridization, Northern blot, Western blot, and RIA analyses. In the rat lung, combination of hypoxia and LPS treatments overcomes ADM induction occurring after each treatment alone. With in situ techniques, the synergistic effect of both stimuli mainly correlates with ADM expression in inflammatory cells within blood vessels and, to a lesser extent, to cells in the lung parenchyma and bronchiolar epithelial cells. In the in vitro model, hypoxia and hypoxia + LPS treatments caused a similar strong induction of ADM expression and secretion in epithelial and endothelial cell lines. In alveolar macrophages, however, LPS-induced ADM expression and secretion were further increased by the concomitant exposure to hypoxia, thus paralleling the in vivo response. In conclusion, ADM expression is highly induced in a variety of key lung cell types in this rat model of ALI by combination of hypoxia and LPS, suggesting an essential role for this mediator in this syndrome.     

Inhibition of glycolysis alleviates lipopolysaccharide-induced acute lung injury in a mouse model

Gluconic metabolic reprogramming, immune response, and inflammation are intimately linked. Glycolysis involves in the pathologic progress in acute and chronic inflammatory diseases. However, the involvement of glycolysis in the acute lung injury (ALI) is still unclear. This study investigated the role of glycolysis in an animal model of ALI. First, we found that lactate content in serum was remarkably increased in ALI patients and a murine model induced by intratracheal administration of lipopolysaccharide (LPS). The key proteins involving in glycolysis were robustly elevated, including HK2, PKM2, and HIF-1α. Intriguingly, inhibition of glycolysis by 2-deoxyglucose (2-DG) pronouncedly attenuated the lung tissue pathological injury, accumulation of neutrophil, oxidative stress, expression of proinflammatory factors in the lung of ALI mice induced by LPS. The 2-DG treatment also strongly suppressed the activation of the NOD-like receptor (NLR) family and pyrin domain-containing protein 3 (NLRP3) inflammasome. Furthermore, we investigated the role of glycolysis in the inflammatory response of primary murine macrophages activated by LPS in vitro. We found that the 2-DG treatment remarkably reduced the expression of proinflammatory factors induced by LPS, including tumor necrosis factor-α messenger RNA (mRNA), pro-interleukin (IL)-1β mRNA, pro-IL-18 mRNA, NLRP3 mRNA, caspase-1 mRNA, and IL-1β protein. Altogether, these data provide a novel link between gluconic metabolism reprogramming and uncontrolled inflammatory response in ALI. This study suggests glycolytic inhibition as an effective anti-inflammatory strategy in treating ALI.     

miR-146b overexpression ameliorates lipopolysaccharide-induced acute lung injury in vivo and in vitro

Acute respiratory distress syndrome (ARDS) is a type of acute lung injury (ALI), which causes high morbidity and mortality. So far, effective clinical treatment of ARDS is still limited. Recently, miR-146b has been reported to play a key role in inflammation. In the present study, we evaluated the functional role of miR-146b in ARDS using the murine model of lipopolysaccharide (LPS)-induced ALI. The miR-146b expression could be induced by LPS stimulation, and miR-146b overexpression was required in the maintenance of body weight and survival of ALI mice; after miR-146b overexpression, LPS-induced lung injury, pulmonary inflammation, total cell and neutrophil counts, proinflammatory cytokines, and chemokines in bronchial alveolar lavage (BAL) fluid were significantly reduced. The promotive effect of LPS on lung permeability through increasing total protein, albumin and IgM in BAL fluid could be partially reversed by miR-146b overexpression. Moreover, in murine alveolar macrophages, miR-146b overexpression reduced LPS-induced TNF-α and interleukin (IL)-1β releasing. Taken together, we demonstrated that miR-146b overexpression could effectively improve the LPS-induced ALI; miR-146b is a promising target in ARDS treatment.     

Transcriptional repression of human cad gene by hypoxia inducible factor-1alpha

De novo biosynthesis of pyrimidine nucleotides provides essential precursors for DNA synthesis and cell proliferation. The first three steps of de novo pyrimidine biosynthesis are catalyzed by a multifunctional enzyme known as CAD (carbamoyl phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase). In this work, a decrease in CAD expression is detected in numerous cell lines and primary culture human stromal cells incubated under hypoxia or desferrioxamine (DFO)-induced HIF-1alpha accumulation. A putative hypoxia response element (HRE) binding matrix is identified by analyzing human cad-gene promoter using a bioinformatic approach. Promoter activity assays, using constructs harboring the cad promoter (-710/+122) and the -67/HRE fragment (25-bases), respectively, demonstrate the suppression of reporter-gene expression under hypoxia. Suppression of cad-promoter activity is substantiated by forced expression of wild-type HIF-1alpha but abolished by overexpression of dominant-negative HIF-1alpha. A chromatin immunoprecipitation assay provides further evidence that HIF-1alpha binds to the cad promoter in vivo. These data demonstrate that the cad-gene expression is repressed by HIF-1alpha, which represents a functional link between hypoxia and cell-cycle arrest.     

Silibinin inhibits expression of HIF-1α through suppression of protein translation in prostate cancer cells

Silibinin is a polyphenolic flavonoid isolated from the milk thistle (Silybum marianum) and is reported to exhibit anticancer properties. Recently, it has been reported that silibinin inhibits hypoxia-inducible factor-1α (HIF-1α) expression in cancer cells. However, the precise mechanism by which silibinin decreases HIF-1 expression is not fully understood. In this study, silibinin inhibited basal and hypoxia induced expression levels of HIF-1α protein in LNCaP and PC-3 prostate cancer cells, while the rate of HIF-1α protein degradation and mRNA levels were not affected. We found that the decrease in HIF-1 protein by silibinin correlated with suppression of de novo synthesis of HIF-1α protein. Silibinin inhibited global protein synthesis coincided with reduction of eIF4F complex formation and induction of phosphorylation of the translation initiation factor 2α (eIF-2α) which can cause inhibition of general protein synthesis. These results suggest that silibinin’s activity to inhibit HIF-1α protein expression is associated with the suppression of global protein translation.     

复方清下汤对脓毒症大鼠细胞因子调控及其肺损伤的作用

目的盲肠结扎穿孔导致大肠埃希菌腹膜炎进而建立脓毒症肺损伤大鼠模型,检测炎性反应时,细胞因子的调控变化,探讨肺水肿的形成机制。经复方清下汤处理后检测上述变化,以期为脓毒症肺损伤的防治提出可能的新途径。方法将健康SD大鼠随机分为4组,每组10只：假手术组（SHAM组）,只翻动盲肠,不做其他处理;脓毒症肺损伤组（模型组）,盲肠结扎穿孔诱发AL（急性肺损伤）I模型;盲肠结扎穿孔＋复方清下汤组（造模后立即灌胃给药,造模后8 h再次灌胃1次,剂量为10 m l/kg）;盲肠结扎穿孔＋头孢哌酮/舒巴坦组（抗生素舒普深）（造模后立即静脉注射1次,造模后8 h再次静脉注射1次,剂量为0.2 g/kg）造模24 h后收集标本。分别观察大鼠的一般状态,肺组织匀浆MPO的测定,留取下腔静脉血清进行TNF-α的测定。镜下观察肺组织病理形态学改变,测量肺湿/干比值的变化。结果与SHAM组比较,模型组MPO、TNF-α水平明显升高（P〈0.01）,肺间质和肺泡内水肿,伴大量红细胞渗出（出血）和纤维素沉积,肺泡间隔毛细血管内皮细胞高度肿胀。肺湿/干比值明显增加（P〈0.01）,抗生素及中药处理组与模型组比较,MPO、TNF-α水平明显降低（P〈0.01）,肺湿/干比值明显降低（P〈0.01）,肺组织镜下表现：中药处理组及抗生素组较模型组肺泡间隔变窄,毛细血管内皮细胞肿胀减轻,出血减轻,纤维素渗出明显减少。结论脓毒症大鼠肺损伤时细胞因子TNF-α过度表达,炎性介质的过度表达可能是造成脓毒症肺损伤的重要原因,而复方清下汤可以减轻脓毒症时的肺损伤和抑制TNF-α的表达,它们之间可能存在一定的联系。     

Serum SP-D is a marker of lung injury in rats

Pulmonary surfactant protein D (SP-D) is expressed in alveolar type II and bronchiolar epithelial cells and is secreted into alveoli and conducting airways. However, SP-D has also been measured in serum and is increased in patients with acute respiratory distress syndrome, pulmonary fibrosis, and alveolar proteinosis. To demonstrate that SP-D can be measured in rat serum, we instilled rats with keratinocyte growth factor, which produces type II cell hyperplasia and an increase in SP-D in bronchoalveolar lavage fluid (BALF). To evaluate serum SP-D as a biomarker of lung injury, we examined several injury models. In rats treated with 1 unit of bleomycin, serum SP-D was elevated on days 3, 7, 14, and 28 after instillation, and SP-D mRNA was increased in focal areas as detected by in situ hybridization. However, there was no increase in whole lung SP-D mRNA when the expression was normalized to whole lung 18S rRNA. After instillation of 2 units of bleomycin, the serum levels of SP-D were higher, and SP-D was also increased in BALF and lung homogenates. In another model of subacute injury, serum SP-D was increased in rats treated with paraquat plus oxygen. Finally to evaluate acute lung injury, we instilled rats with HCl; SP-D was increased at 4 h after instillation. Our data indicate that serum SP-D may be a useful indicator of lung injury and type II cell hyperplasia in rats.     

Elevation of KL-6, a lung epithelial cell marker,in plasma and epithelial lining fluid in acute respiratory distress syndrome

KL-6 is a pulmonary epithelial mucin more prominently expressed on the surface membrane of alveolar type II cells when these cells are proliferating, stimulated, and/or injured. We hypothesized that high levels of KL-6 in epithelial lining fluid and plasma would reflect the severity of lung injury in patients with acute lung injury (ALI). Epithelial lining fluid was obtained at onset (day 0) and day 1 of acute respiratory distress syndrome (ARDS)/ALI by bronchoscopic microsampling procedure in 35 patients. On day 0, KL-6 and albumin concentrations in epithelial lining fluid were significantly higher than in normal controls (P < 0.001), and the concentrations of KL-6 in epithelial lining fluid (P < 0.002) and in plasma (P < 0.0001) were higher in nonsurvivors than in survivors of ALI/ARDS. These observations were corroborated by the immunohistochemical localization of KL-6 protein expression in the lungs of nonsurvivors with ALI and KL-6 secretion from cultured human alveolar type II cells stimulated by proinflammatory cytokines. Because injury to distal lung epithelial cells, including alveolar type II cells, is important in the pathogenesis of ALI, the elevation of KL-6 concentrations in plasma and epithelial lining fluid could be valuable indicators for poor prognosis in clinical ALI.     

cAMP-dependent protein kinase is essential for hypoxia-mediated epithelial–mesenchymal transition,migration, and invasion in lung cancer cells

Lung cancer is the leading cause of cancer-related death worldwide. Hypoxia is known to increase cancer cell migration and invasion. We have previously reported that hypoxia induces epithelial–mesenchymal transition (EMT) in lung cancer cells. However, it is unknown whether hypoxia promotes lung cancer cell migration and invasion via EMT and whether cyclic AMP (cAMP) dependent protein kinase (PKA) plays a role in this process. We found that hypoxia increased PKA activity and induced mRNA and protein expression of PKA catalytic subunit α (PKACA), and regulatory subunits R1A and R1B. Knockdown of HIF-1/2α prevented hypoxia-mediated induction of PKACA mRNA expression and PKA activity. Inhibition of PKA activity with chemical inhibitors prevented EMT induced by hypoxia and tumor growth factor β1. However, activation of PKA by forskolin and 8-Br-cAMP did not induce EMT. Furthermore, treatment with H89 and knockdown of PKACA prevented hypoxia-mediated, EMT, cell migration, and invasion, whereas overexpression of mouse PKACA rescued hypoxia-mediated migration and invasion in PKACA deficient cancer cells. Our results suggest that hypoxia enhances PKA activity by upregulating PKA gene expression in a HIF dependent mechanism and that PKA plays a key role in hypoxia-mediated EMT, migration, and invasion in lung cancer cells.     

雌孕激素对ALI大鼠α-ENaC、BNP、TNF-α表达的影响

目的:研究雌孕激素对ALI大鼠II型肺泡上皮细胞钠通道(epithelial Na+channel,ENaC)α亚基,血清脑钠肽(brain natriuretic peptide,BNP)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)表达的影响,探讨肺泡内液体清除的机制。方法:雌性未成年SD大鼠30只,随机分为3组:对照组(静脉注入生理盐水8 mL/kg)、ALI模型组(静脉注入油酸0.1 mL/kg)、雌孕激素联合组(在油酸注入前12 h、36 h皮下注射1∶1 000雌孕激素混合液0.1 mL)。建模后6 h处死大鼠,观察肺组织病理改变,测定肺湿重/干重比值、肺系数,肺组织匀浆中α-ENaC mRNA及蛋白的表达及血清BNP、TNF-α的表达。结果:与对照组比较,模型组出现明显肺水肿、出血及炎性细胞浸润,肺湿重/干重比值、肺系数及血清中BNP、TNF-α表达增加,而α-ENaC mRNA及蛋白明显降低(P<0.01);雌孕激素联合组与ALI模型组比较,肺水肿、出血及炎性细胞浸润要轻,肺湿重/干重比值、肺系数、BNP下降,α-ENaC mRNA及蛋白增加(P<0.05)。结论:雌孕激素联合治疗可上调α-ENaC mRNA及蛋白的表达,减少BNP表达,促进肺泡内液体的清除,为ALI/急性呼吸窘迫综合症(ARDS)的治疗和监控提供新的方法。     

Hypoxia-inducible factor plays a gut-injurious role in intestinal ischemia reperfusion injury

Gut injury and loss of normal intestinal barrier function are key elements in the paradigm of gut-origin systemic inflammatory response syndrome, acute lung injury, and multiple organ dysfunction syndrome (MODS). As hypoxia-inducible factor (HIF-1) is a critical determinant of the physiological and pathophysiological response to hypoxia and ischemia, we asked whether HIF-1 plays a proximal role in the induction of gut injury and subsequent lung injury. Using partially HIF-1α-deficient mice in an isolated superior mesenteric artery occlusion (SMAO) intestinal ischemia reperfusion (I/R) injury model (45 min SMAO followed by 3 h of reperfusion), we showed a direct relationship between HIF-1 activation and intestinal I/R injury. Specifically, partial HIF-1α deficiency attenuated SMAO-induced increases in intestinal permeability, lipid peroxidation, mucosal caspase-3 activity, and IL-1β mRNA levels. Furthermore, partial HIF-1α deficiency prevented the induction of ileal mucosal inducible nitric oxide synthase (iNOS) protein levels after SMAO and iNOS deficiency ameliorated SMAO-induced villus injury. Resistance to SMAO-induced gut injury was also associated with resistance to lung injury, as reflected by decreased levels of myeloperoxidase, IL-6 and IL-10 in the lungs of HIF-1α(+/-) mice. In contrast, a short duration of SMAO (15 min) followed by 3 h of reperfusion neither induced mucosal HIF-1α protein levels nor caused significant gut and lung injury in wild-type or HIF-1α(+/-) mice. This study indicates that intestinal HIF-1 activation is a proximal regulator of I/R-induced gut mucosal injury and gut-induced lung injury. However, the duration and severity of the gut I/R insult dictate whether HIF-1 plays a gut-protective or deleterious role.     

Effect of intermittent hypoxia or hyperoxia on lung development in preterm rat neonates during constant oxygen therapy

Impaired lung development is a major negative factor in the survival of preterm neonates. The present study was aimed to investigate the impact of constant oxygen, intermittent hyperoxia, and hypoxia on the lung development in preterm rat neonates. Neonatal rats were exposed to 40% O₂ with or without brief hyperoxia episodes (95% O₂) or brief hypoxia episodes (10% O₂) from day 0 to day 14, or to room air. The body weight, radical alveolar count (RAC), and total antioxidant capacity (TAOC) were significantly lower whereas the lung coefficient and malondialdehyde (MDA) were significantly higher in the hyperoxia and hypoxia groups than the air control and constant oxygen group at day 7, day 14, and day 21 after birth. The lung function indexes were reduced by intermittent hyperoxia and hypoxia. In contrast, the constant oxygen therapy increased the lung function. HIF-1α and VEGF expression were significantly increased by hypoxia and decreased by hyperoxia. The constant oxygen therapy only decreased the HIF-1α expression at day 14 and 21. In summary, the constant oxygen treatment promoted lung function without affecting the antioxidative capacity in preterm rat neonates. While intermittent hyperoxia and hypoxia inhibited lung development, decreased antioxidative capacity, and dysregulated HIF-1α/VEGF signaling in preterm rat neonates.     

Nuclear import of aristaless-related homeobox protein via its NLS1 regulates its transcriptional function

Migration and invasion of fibroblast-like synoviocytes (FLSs) are critical in the pathogenesis of rheumatoid arthritis (RA). Hypoxic conditions are present in RA joints, and hypoxia has been extensively studied in angiogenesis and inflammation. However, its effect on the migration and invasion of RA-FLSs remains unknown. In this study, we observed that RA-FLSs exposed to hypoxic conditions experienced epithelial–mesenchymal transition (EMT), with increased cell migration and invasion. We demonstrated that hypoxia-induced EMT was accompanied by increased hypoxia-inducible factor (HIF)-1α expression and activation of Akt. After knockdown or inhibition of HIF-1α in hypoxia by small interfering RNA or genistein (Gen) treatment, the EMT transformation and invasion ability of FLSs were regained. HIF-1α could be blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, indicating that HIF-1α activation was regulated by the PI3K/Akt pathway. Administration of LY294002 (20 mg/kg, intra-peritoneally) twice weekly and Gen (25 mg/kg, by gavage) daily for 3 weeks from day 20 after primary immunization in a collagen-induced arthritis rat model, markedly alleviated the clinical signs, radiology progression, synovial hyperplasia, and inflammatory cells infiltration of joints. Thus, results of this study suggest that activation of the PI3K/Akt/HIF-1α pathway plays a pivotal role in mediating hypoxia-induced EMT transformation and invasion of RA-FLSs under hypoxia.     

The microRNA miR-17 regulates lung FoxA1 expression during lipopolysaccharide-induced acute lung injury

Acute lung injury (ALI) is a severe pulmonary disease that causes a high number of fatalities worldwide. Studies have shown that FoxA1 expression is upregulated during ALI and may play an important role in ALI by promoting the apoptosis of alveolar type II epithelial cells. However, the mechanism of FoxA1 overexpression in ALI is unclear. In this study, an in vivo murine model of ALI and alveolar type II epithelial cells injury was induced using lipopolysaccharide (LPS). LPS upregulated FoxA1 in the lung tissue of the in vivo ALI model and in LPS-challenged type II epithelial cells. In contrast, miR-17 was significantly downregulated in these models. After miR-17 antagomir injection, the expression of FoxA1 was significantly increased in ALI mice. MiR-17 mimics could significantly inhibit FoxA1 mRNA and protein expression, whereas the miR-17 inhibitor could significantly increase FoxA1 mRNA and protein expression in LPS-induced type II epithelial cells. Thus, our results suggest that the downregulation of miR-17 expression could lead to FoxA1 overexpression in ALI.