Cell‐specific elevation of NRF2 and sulfiredoxin‐1 as markers of oxidative stress in the lungs of idiopathic pulmonary fibrosis and non‐specific interstitial pneumonia

Mazur W, Lindholm P, Vuorinen K, Myllärniemi M, Salmenkivi K, Kinnula VL. Cell‐specific elevation of NRF2 and sulfiredoxin‐1 as markers of oxidative stress in the lungs of idiopathic pulmonary fibrosis and non‐specific interstitial pneumonia. APMIS 2010; 118: 703–12. Human idiopathic pulmonary fibrosis (IPF) and non‐specific interstitial pneumonia (NSIP) have been proposed to be attributable to oxidative stress. The nuclear factor, erythroid derived 2, like protein (NRF2)–sulfiredoxin‐1 (SRX1) pathway was hypothesized to be associated with the pathogenesis of human pulmonary fibrosis. Several methods including digital morphometry were used in the assessment of the cell‐specific localization and expression of NRF2 and SRX1 and selected proteins linked to their activation/stability in human IPF/usual interstitial pneumonia (UIP) and NSIP lung. The proteins of the NRF2 pathway were localized in the hyperplastic alveolar epithelium and inflammatory cells in IPF and NSIP, but were absent in the fibroblastic foci characteristic of IPF. Morphometric evaluation revealed NRF2 and KEAP1 to be significantly elevated in the hyperplastic alveolar epithelium compared with the normal alveolar epithelium, and NRF2 was remarkably expressed in the nuclear compartment of the hyperplastic cells. SRX1 was expressed mainly in alveolar macrophages, and the number of SRX1‐positive macrophages/surface area was elevated in NSIP, a disease which contains more marked inflammatory reaction compared with the IPF/UIP lung. The expression of the NRF2 pathway in human IPF and NSIP is further evidence that the pathogenesis of human fibrotic lung diseases is oxidant‐mediated and originates from the alveolar epithelium.     

Hirsutella sinensis mycelium regulates autophagy of alveolar macrophages via TLR4/NF-κB signaling pathway

Background: Hirsutella sinensis mycelium (HSM) has potent anti-pulmonary fibrotic activities and has been proposed as an effective treatment for idiopathic pulmonary fibrosis. Macrophages are the main innate immune cells in the lung tissue, playing key roles in pulmonary fibrosis repair and homeostasis. Excessive macrophage autophagy plays a vital role in pulmonary fibrosis. The protective effect of HSM on macrophages of bleomycin (BLM)-induced pulmonary fibrotic mice remain unclear.Methods: In this study, we collected lung tissue and bronchoalveolar lavage fluid (BALF) samples from pulmonary fibrotic mice. Meanwhile, alveolar macrophages were isolated and murine macrophage RAW264.7 cell line was cultured for further study of HSM autophagy.Results: First, we found that HSM decreased the number of autophagosomes, as well as the levels of LC3B and ATG5, and increased the protein level of P62 during the development of pulmonary fibrosis. Meanwhile, HSM reduced alveolar macrophages infiltration into the BALF and inhibited their accumulation in the fibrotic lung tissue. Flow cytometry analysis showed that HSM administration inhibited the autophagy marker LC3B expression in CD11b^loCD11c^hi alveolar macrophages in BLM-induced lung fibrosis without affecting CD11b^hiCD11c^lo interstitial macrophages. Transmission electron microscopy and JC-1 staining for mitochondrial membrane potential of alveolar macrophages also verified that the HSM significantly decreased autophagy in the alveolar macrophages of BLM-treated mice. In vitro, autophagosomes-lysosome fusion inhibitor chloroquine (CQ) was pre-incubated with RAW264.7 cells, and HSM reduced CQ-induced autophagosomes accumulation. TLR4 signaling inhibitor CLI095 reversed the above effects, suggesting HSM could reduce the cumulation of autophagosomes dependent on TLR4. Furthermore, lipopolysaccharide (LPS)-stimulated TLR4-related autophagy was significantly inhibited by HSM treatment. In addition, the protein expressions of TLR4 and phospho-NF-κB p65 were markedly inhibited in cells treated with HSM.Conclusions: These results indicated that HSM could inhibit the autophagy of alveolar macrophages through TLR4/NF-κB signaling pathway to achieve anti-fibrotic effect.     

Didecyldimethylammonium chloride induces pulmonary fibrosis in association with TGF-β signaling in mice

Didecyldimethylammonium chloride (DDAC) is a representative dialkyl-quaternary ammonium compound that is used as a disinfectant against several pathogens and is also used in commercial, industrial, and residential settings. We previously investigated toxicity on air way system following single instillation of DDAC to the lungs in mice, and found that DDAC causes pulmonary injury, which is associated with altered antioxidant antimicrobial responses; the inflammatory phase is accompanied or followed by fibrotic response. The present study was conducted to monitor transforming growth factor-β (TGF-β) signaling in pulmonary fibrosis induced by DDAC. Mice were intratracheally instilled with DDAC and sacrificed 1, 3, or 7 days after treatment to measure TGF-β signaling. In order to further evaluate TGF-β signaling, we treated isolated mouse lung fibroblasts with DDAC. Fibrotic foci were observed in the lungs on day 3, and were widely extended on day 7, with evidence of increased α-smooth muscle actin-positive mesenchymal cells and upregulation of Type I procollagen mRNA. Developing fibrotic foci were likely associated with increased expression of Tgf-β1 mRNA, in addition to decreased expression of Bone morphogenetic protein-7 mRNA. In fibrotic lung samples, the expression of phosphorylated SMAD2/3 was considerably higher than that of phosphorylated SMAD1/5. In isolated lung fibroblasts, the mRNA levels of Tgf-β1 were specifically increased by DDAC treatment, which prolonged phosphorylation of SMAD2/3. These effects were abolished by treatment with SD208 – a TGF-βRI kinase inhibitor. The results suggest that DDAC induces pulmonary fibrosis in association with TGF-β signaling.     

Protective roles of pulmonary rehabilitation mixture in experimental pulmonary fibrosis in vitro and in vivo

Abnormal high mobility group protein B1 (HMGB1) activation is involved in the pathogenesis of pulmonary fibrosis. Pulmonary rehabilitation mixture (PRM), which combines extracts from eight traditional Chinese medicines, has very good lung protection in clinical use. However, it is not known if PRM has anti-fibrotic activity. In this study, we investigated the effects of PRM on transforming growth factor-β1 (TGF-β1)-mediated and bleomycin (BLM)-induced pulmonary fibrosis in vitro and in vivo. The effects of PRM on TGF-β1-mediated epithelial-mesenchymal transition (EMT) in A549 cells, on the proliferation of human lung fibroblasts (HLF-1) in vitro, and on BLM-induced pulmonary fibrosis in vivo were investigated. PRM treatment resulted in a reduction of EMT in A549 cells that was associated with attenuating an increase of vimentin and a decrease of E-cadherin. PRM inhibited the proliferation of HLF-1 at an IC₅₀ of 0.51 µg/mL. PRM ameliorated BLM-induced pulmonary fibrosis in rats, with reduction of histopathological scores and collagen deposition, and a decrease in α-smooth muscle actin (α-SMA) and HMGB1 expression. An increase in receptor for advanced glycation end-product (RAGE) expression was found in BLM-instilled lungs. PRM significantly decreased EMT and prevented pulmonary fibrosis through decreasing HMGB1 and regulating RAGE in vitro and in vivo. PRM inhibited TGF-β1-induced EMT via decreased HMGB1 and vimentin and increased RAGE and E-cadherin levels. In summary, PRM prevented experimental pulmonary fibrosis by modulating the HMGB1/RAGE pathway.     

Critical role for iron accumulation in the pathogenesis of fibrotic lung disease

Increased iron levels and dysregulated iron homeostasis, or both, occur in several lung diseases. Here, the effects of iron accumulation on the pathogenesis of pulmonary fibrosis and associated lung function decline was investigated using a combination of murine models of iron overload and bleomycin-induced pulmonary fibrosis, primary human lung fibroblasts treated with iron, and histological samples from patients with or without idiopathic pulmonary fibrosis (IPF). Iron levels are significantly increased in iron overloaded transferrin receptor 2 (Tfr2) mutant mice and homeostatic iron regulator (Hfe) gene–deficient mice and this is associated with increases in airway fibrosis and reduced lung function. Furthermore, fibrosis and lung function decline are associated with pulmonary iron accumulation in bleomycin-induced pulmonary fibrosis. In addition, we show that iron accumulation is increased in lung sections from patients with IPF and that human lung fibroblasts show greater proliferation and cytokine and extracellular matrix responses when exposed to increased iron levels. Significantly, we show that intranasal treatment with the iron chelator, deferoxamine (DFO), from the time when pulmonary iron levels accumulate, prevents airway fibrosis and decline in lung function in experimental pulmonary fibrosis. Pulmonary fibrosis is associated with an increase in Tfr1⁺ macrophages that display altered phenotype in disease, and DFO treatment modified the abundance of these cells. These experimental and clinical data demonstrate that increased accumulation of pulmonary iron plays a key role in the pathogenesis of pulmonary fibrosis and lung function decline. Furthermore, these data highlight the potential for the therapeutic targeting of increased pulmonary iron in the treatment of fibrotic lung diseases such as IPF. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Epithelial cell apoptosis by fas ligand-positive myofibroblasts in lung fibrosis

The Fas/Fas ligand (FasL) apoptotic pathway has been shown to be involved in bleomycin-induced lung fibrosis. We examined the hypothesis that myofibroblasts from fibrotic lungs possess a cytotoxic phenotype that causes apoptosis of epithelial cells via the Fas/FasL pathway. We show in vivo epithelial cell apoptosis and associated upregulation of Fas and apoptotic Fas pathway genes in epithelial cells of lungs with bleomycin-induced fibrosis. In addition, we show that FasL surface molecules are overexpressed on alpha-SMA-positive cells in mice with bleomycin-induced fibrosis, and in humans with idiopathic pulmonary fibrosis. This enables the molecules to kill Fas-positive epithelial cells. In contrast, FasL-deficient myofibroblasts lose this myofibroblast cytotoxic phenotype, both in vivo and in vitro. In vivo, there was no bleomycin-induced epithelial cell apoptosis, as assessed by specific M30 staining in chimeric FasL-deficient mice that lacked FasL-positive myofibroblasts. In vitro, FasL-positive, but not FasL-negative myofibroblasts, induce mouse lung epithelial cell apoptosis. Thus myofibroblast cytotoxicity may underlie the absence of re-epithelialization, resulting in persistent lung fibrosis.     

Extracellular superoxide dismutase in pulmonary fibrosis

Disruption of the oxidant/antioxidant balance in the lung is thought to be a key step in the development of many airway pathologies. Hence, antioxidant enzymes play key roles in controlling or preventing pulmonary diseases related to oxidative stress. The superoxide dismutases (SOD) are a family of enzymes that play a pivotal role protecting tissues from damage by oxidant stress by scavenging superoxide anion, which prevents the formation of other more potent oxidants such as peroxynitrite and hydroxyl radical. Extracellular SOD (EC-SOD) is found predominantly in the extracellular matrix of tissues and is ideally situated to prevent cell and tissue damage initiated by extracellularly produced ROS. EC-SOD has been shown to be protective in several models of interstitial lung disease, including pulmonary fibrosis. In addition, alterations in EC-SOD expression are also present in human idiopathic pulmonary fibrosis (IPF). This review discusses EC-SOD regulation in response to pulmonary fibrosis in animals and humans and reviews possible mechanisms by which EC-SOD may protect against fibrosis.     

Alcohol dehydrogenase 3 contributes to the protection of liver from nonalcoholic steatohepatitis

Nutritional steatohepatitis is closely associated with dysregulation of lipid metabolism and oxidative stress control. ADH3 is a highly conserved bifunctional enzyme involved in formaldehyde detoxification and termination of nitric oxide signaling. Formaldehyde and nitric oxide are nonenzymatically conjugated with glutathione, which is regenerated after ADH3 metabolizes the conjugates. To clarify roles of ADH3 in nutritional liver diseases, we placed Adh3‐null mice on a methionine‐ and choline‐deficient (MCD) diet. The Adh3‐null mice developed steatohepatitis more rapidly than wild‐type mice, indicating that ADH3 protects liver from nutritional steatohepatitis. NRF2, which is a key regulator of cytoprotective genes against oxidative stress, was activated in the Adh3‐null mice with liver damage. In the absence of NRF2, the Adh3 disruption caused severe steatohepatitis by the MCD diet feeding accompanied by significant decrease in glutathione, suggesting cooperative function between ADH3 and NRF2 in the maintenance of cellular glutathione level for cytoprotection. Conversely, with enhanced NRF2 activity, the Adh3 disruption did not cause steatohepatitis but induced steatosis, suggesting that perturbation of lipid metabolism in ADH3‐deficiency is not compensated by NRF2. Thus, ADH3 protects liver from steatosis by supporting normal lipid metabolism and prevents progression of steatosis into steatohepatitis by maintaining the cellular glutathione level.     

Bone marrow mesenchymal stem cells protect against bleomycin-induced pulmonary fibrosis in rat by activating Nrf2 signaling

Pulmonary fibrosis is a progressive and lethal disorder. Although the precise mechanisms of pulmonary fibrosis are not fully understood, oxidant/antioxidant may play an important role in many of the processes of inflammation and fibrosis. Keap1-Nrf2-ARE pathway represents one of the most important cellular defense mechanisms against oxidative stress. Mesenchymal stem cells (MSC) are in clinical trials for widespread indications including musculoskeletal, neurological, cardiac and haematological disorders. One emerging concept is that MSCs may have paracrine, rather than a functional, roles in lung injury repair and regeneration. In the present study, we investigated bone marrow mesenchymal stem cells (BMSCs) for the treatment of bleomycin-induced pulmonary fibrosis. Our results showed that BMSCs administration significantly ameliorated the bleomycin mediated histological alterations and blocked collagen deposition with parallel reduction in the hydroxyproline level. The gene expression levels of NAD(P)H: quinine oxidoreductase 1 (NQO1), gama-glutamylcysteine synthetase (γ-GCS), heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2), attenuated by bleomycin, were increased up to basal levels after BMSCs transplantation. BMSCs significantly increased superoxide dismutase (SOD) activity and inhibited malondialdehyde (MDA) production in the injured lung. The present study provides evidence that BMSCs may be a potential therapeutic reagent for the treatment of lung fibrosis.     

Genetic polymorphism and antioxidant activity in interventions of tobacco-related diseases of the abdominal aorta

IntroductionThe aim of this study was to determine the genetic polymorphisms of some antioxidant enzymes together with oxidative stress and the response of some antioxidant enzymes against this situation in vascular and endovascular interventions performed for diseases of the infrarenal abdominal aorta.Material and methodsTwenty-four current or ex-smoker patients (eight aortoiliac occlusive disease (AOD), 16 abdominal aortic aneurysm (AAA)) who were operated were included in this pilot study. Malonyl dialdehyde (MDA) levels, as an indicator of oxidative stress, reduced glutathione, catalase and superoxide dismutase enzymes, which are indicators of antioxidant status, which were measured in aortofemoral bypass in AODs, and in endovascular abdominal aortic aneurysms repairs in the preoperative, operative, and postoperative periods. Genetic polymorphisms of these antioxidant enzymes developing a response to the damage in the preoperative blood samples were determined by using the PCR-RFLP method.ResultsThe lack of a significant increase of MDA (nmol/ml preoperative: 2.57 ±0.19, operative: 2.59 ±0.21, postoperative: 2.64 ±0.22, p = 0.63) in the oxidative damage in the operative and postoperative periods compared to the preoperative period prevented the damage and was thought to be associated with the elevation of some effective antioxidant parameters in the operative and postoperative periods.ConclusionsIt may be thought that both types of interventions are quite reliable in terms of oxidative damage and, accordingly, the effect of the procedure-associated oxidative damage in the postoperative complications is low or ineffective. Two genotypes were obtained in each of the three gene areas of the patients, and no statistical significance was determined between the genotypes (p > 0.05).     

Indices of oxidative stress in hamster lung following exposure to cobalt(II) ions: in vivo and in vitro studies

Cobalt, a metal with numerous industrial applications, has been associated with lung disease, an extreme form of which is an interstitial fibrosis. The biochemical mechanisms underlying this toxicity are not understood. In vitro studies have suggested that cobalt(II) ions are able to generate reactive oxidant species (possibly hydroxyl radical) in a reaction with hydrogen peroxide, and we have hypothesized that the occurrence of such an event in lung tissue, and the subsequent development of oxidative damage, may contribute to this pulmonary toxicity. The intratracheal instillation of CoCl2 into hamster lungs resulted after 3 h in decreased levels of reduced glutathione and increases in levels of oxidized glutathione and in the activity of the pentose phosphate pathway. These changes, which are compatible with the generation of oxidative stress, were reversed by 48 h at low Co2+ doses (1.0 to 1,000 micrograms/kg). Irreversible changes at higher doses coincided with the onset of pulmonary edema. Incubation of lung slices with CoCl2 (0.1 to 10 mM) resulted in time- and Co2+ concentration-dependent increases in levels of oxidized glutathione and protein-mixed disulfides and a decrease in reduced glutathione. A concentration-dependent stimulation of the pentose phosphate pathway was also observed. These changes preceded the detection of overt cell toxicity, as assessed by various biochemical parameters. These data indicate that thiol oxidation constitutes an early event in the pulmonary toxicity of cobalt(II) ions and are compatible with the hypothesis that the generation of oxidative stress may be of significance to the toxic process.     

Decreased prostaglandin E2 synthesis by lung fibroblasts isolated from rats with bleomycin-induced lung fibrosis

In order to clarify the mechanism of pulmonary fibrosis, we examined the functional changes of lung fibroblasts in bleomycin (BLM)-induced pulmonary fibrosis. Lung fibroblastic cells were obtained from rat lungs after an intratracheal treatment of BLM or saline. The spontaneous proliferation of BLM-treated rat fibroblasts (BRF), which was estimated by 3H-TdR incorporation and direct cell counting, was significantly more rapid than that of normal saline-treated rat fibroblasts (NRF). Next, we investigated prostaglandin (PG) E2 synthesis by BRF and NRF, with or without stimulation by interleukin (IL)-1 alpha, and found that PGE2 production by BRF was significantly less than that by NRF. There was no significant difference in cyclooxygenase (COX) activity and COX-2 mRNA level between BRF and NRF, indicating that the change in PGE2 production was independent of COX, a rate-limiting enzyme for the production of PGE2. These results suggest that the proliferation of fibroblasts is down-regulated by PGE2 released from themselves in normal lungs in an autocrine fashion, thus the decreased PGE2 production observed in lung fibroblasts from rats with BLM-induced pulmonary fibrosis may result in the excessive fibroblast proliferation in this disorder. Overall, these findings throw some light on the mechanism of development of BLM-induced pulmonary fibrosis.     

Immune Complex Injury of the Lung

Abundant evidence currently exists to suggest that immune complexes play an important role in inflammatory diseases of the lung. Clinically, idiopathic pulmonary fibrosis, eosinophilic granuloma of lung, and systemic lupus erythematosus have been shown to be associated with the presence of immune complexes both in lung and in the serum. Experimentally, there is compelling evidence that acute lung injury can be triggered by the deposition of complexes in vascular walls or by the presence of performed immune complexes instilled into the airways. The observed reactions are, as expected, complement- and neutrophil-dependent. The morphologic changes in lung caused by products of complement activation (C5a and related peptides) depend on whether complement activation occurs within the vasculature or within the airways. Airway activation is associated with intraalveolar accumulations of neutrophils, while intravascular activation leads to intracapillary sequestration of neutrophils. The chronic formation of immune complexes within the vasculature (in the model of “chronic serum sickness”) leads to an interstitial fibrotic reaction and a thickening of basement membranes. Recent studies of intravascularly infused preformed immune complexes indicate a proclivity for certain types of complexes to localize within lung. These “lung-seeking” complexes differ from non-lung-seeking complexes only in the ratio of antigen to antibody. Complement does not seem to alter the tendancy for certain complexes to localize within lung. These studies emphasize the potential importance of immune complexes in lung injury and point out the variety of mechanisms involved in both the localization process and the injury process.