Bleomycin-induced Lung Injury Research Articles

P219 Bleomycin-induced lung injury in testicular cancer patients – results from long-term follow-up

P219 Bleomycin-induced lung injury in testicular cancer patients – results from long-term follow-up

Engraftment of wild-type alveolar type II epithelial cells in surfactant protein C deficient mice.

Childhood interstitial lung disease (chILD) secondary to pulmonary surfactant deficiency is a devastating chronic lung disease in children. Clinical presentation includes mild to severe respiratory failure and fibrosis. There is no specific treatment, except lung transplantation, which is hampered by a severe shortage of donor organs, especially for young patients. Repair of lungs with chILD represents a longstanding therapeutic challenge but cell therapy is a promising strategy. As surfactant is produced by alveolar type II epithelial (ATII) cells, engraftment with normal or gene-corrected ATII cells might provide an avenue to cure. Here we used a chILD disease-like model, Sftpc -/- mice, to provide proof-of-principle for this approach. Sftpc -/- mice developed chronic interstitial lung disease with age and were hypersensitive to bleomycin. We could engraft wild-type ATII cells after low dose bleomycin conditioning. Transplanted ATII cells produced mature SPC and attenuated bleomycin-induced lung injury up to two months post-transplant. This study demonstrates that partial replacement of mutant ATII cells can promote lung repair in a mouse model of chILD-like disease.

Ablation of LRP6 in alpha-smooth muscle actin-expressing cells abrogates lung inflammation and fibrosis upon bleomycin-induced lung injury.

Low-density lipoprotein receptor-related protein 6 (LRP6) is a receptor for Wnt ligands. Tissue fibrosis is a progressive pathological process with excessive extracellular matrix proteins (ECM) deposition. Myofibroblasts, identified by alpha-smooth muscle actin (αSMA) expression, play an important role in tissue fibrosis by producing ECM production. Here we found that Wnt antagonist Dickkopf1 (DKK1) induced gene expressions associated with inflammation and fibrosis in lung fibroblasts. We demonstrated that genetic deletion of LRP6 in αSMA-expressing cells using Acta2 -cre Lrp6 fl/fl ( Lrp6 AKO ) mice abrogated bleomycin (BLM)-induced lung inflammation and fibrosis phenotype, suggesting an important role of LRP6 in modulating inflammation and fibrotic processes in the lung. Our results highlight the crucial role of LRP6 in fibroblasts in regulating inflammation and fibrosis upon BLM-induced lung injury.

Down-regulation of CYTL1 attenuates bleomycin-induced pulmonary fibrosis in mice by inhibiting M2 macrophage polarization via the TGF-β/CCN2 axis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by chronic inflammation, lung tissue fibrotic changes and impaired lung function. Pulmonary fibrosis 's pathological process is thought to be influenced by macrophage-associated phenotypes. IPF treatment requires specific targets that target macrophage polarization. Cytokine-like 1(CYTL1) is a secreted protein with multiple biological functions first discovered in CD34+ haematopoietic cells. However, its possible effects on IPF progression remain unclear. This study investigated the role of CYTL1 in IPF progression in a bleomycin-induced lung injury and fibrosis model. In bleomycin-induced mice, CYTL1 is highly expressed. Moreover, CYTL1 ablation alleviates lung injury and fibrosis in vivo. Further, downregulating CYTL1 reduces macrophage M2 polarization. Mechanically, CYTL1 regulates transforming growth factor β (TGF-β)/connective tissue growth factor (CCN2) axis and inhibition of TGF-β pathway alleviates bleomycin-induced lung injury and fibrosis. In conclusion, highly expressed CYTL1 inhibits macrophage M2 polarization by regulating TGF-β/CCN2 expression, alleviating bleomycin-induced lung injury and fibrosis. CYTL1 could, therefore, serve as a promising IPF target.

Mechanisms of Bleomycin-induced Lung Fibrosis: A Review of Therapeutic Targets and Approaches.

Pulmonary toxicity is a serious side effect of some specific anticancer drugs. Bleomycin is a well-known anticancer drug that triggers severe reactions in the lungs. It is an approved drug that may be prescribed for the treatment of testicular cancers, Hodgkin's and non-Hodgkin's lymphomas, ovarian cancer, head and neck cancers, and cervical cancer. A large number of experimental studies and clinical findings show that bleomycin can concentrate in lung tissue, leading to massive oxidative stress, alveolar epithelial cell death, the proliferation of fibroblasts, and finally the infiltration of immune cells. Chronic release of pro-inflammatory and pro-fibrotic molecules by immune cells and fibroblasts leads to pneumonitis and fibrosis. Both fibrosis and pneumonitis are serious concerns for patients who receive bleomycin and may lead to death. Therefore, the management of lung toxicity following cancer therapy with bleomycin is a critical issue. This review explains the cellular and molecular mechanisms of pulmonary injury following treatment with bleomycin. Furthermore, we review therapeutic targets and possible promising strategies for ameliorating bleomycin-induced lung injury.

Immune response regulation by transduced mesenchymal stem cells with decorin gene on bleomycin-induced lung injury, fibrosis, and inflammation.

Pulmonary fibrosis is a pathological hallmark of lung injury. It is an aggressive disease that replaces normal lung parenchyma by fibrotic tissue. The transforming growth factor-beta-mothers against decapentaplegic homolog 3 (TGF-β1-Smad3) signaling pathway plays a key role in regulating lung fibrosis. Decorin (DCN), a small leucine-rich proteoglycan, has a modulatory effect on the immune system by reversibly binding with TGF-β and reducing its bioavailability. Mesenchymal stem cell (MSC) therapy is a new strategy that has an immune-modulatory capacity. The aim of this study was to introduce a new therapeutic approach to harness remodeling in injured lung. Bone marrow MSCs were isolated and transduced by decorin gene. Lung injury was induced by bleomycin and mice were treated with MSCs, MSCs-decorin, and decorin. Then, oxidative stress biomarkers, remodeling biomarkers, bronchoalveolar lavage cells, and histopathology study were conducted. Reduced catalase and superoxide dismutase increased due to treatments. Elevated malondialdehyde, hydroxyproline, TGF-β levels, and polymorphonuclear cells count decreased in the treated groups. Additionally, the histopathology of lung tissues showed controlled inflammation and fibrosis. Transfected decorin gene to MSCs and used cell therapy could control remodeling and bleomycin-induced lung injury.

Human Muse cells isolated from preterm- and term-umbilical cord delivered therapeutic effects in rat bleomycin-induced lung injury model without immunosuppressant

BackgroundBleomycin (BLM)-induced lung injury is characterized by mixed histopathologic changes with inflammation and fibrosis, such as observed in human patients with bronchopulmonary dysplasia, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease. Although no curative therapies for these lung diseases exist, stem cell therapy has emerged as a potential therapeutic option. Multilineage-differentiating stress-enduring (Muse) cells are endogenous pluripotent- and macrophage-like stem cells distributed in various adult and fetal tissues as stage-specific embryonic antigen-3-positive cells. They selectively home to damaged tissue by sensing sphingosine-1-phosphate and replace the damaged/apoptotic cells by in vivo differentiation. Clinical trials for some human diseases suggest the safety and therapeutic efficacy of intravenously injected human leukocyte antigen-mismatched allogenic Muse cells from adult bone marrow (BM) without immunosuppressant. Here, we evaluated the therapeutic effects of human Muse cells from preterm and term umbilical cord (UC), and adult BM in a rat BLM-induced lung injury model.MethodsRats were endotracheally administered BLM to induce lung injury on day 0. On day 3, human preterm UC-Muse, term UC-Muse, or adult BM-Muse cells were administered intravenously without immunosuppressants, and rats were subjected to histopathologic analysis on day 21. Body weight, serum surfactant protein D (SP-D) levels, and oxygen saturation (SpO2) were monitored. Histopathologic lung injury scoring by the Ashcroft and modified American Thoracic Society document scales, quantitative characterization of engrafted Muse cells, RNA sequencing analysis, and in vitro migration assay of infused Muse cells were performed.ResultsRats administered preterm- and term-UC-Muse cells exhibited a significantly better recovery based on weight loss, serum SP-D levels, SpO2, and histopathologic lung injury scores, and a significantly higher rate of both Muse cell homing to the lung and alveolar marker expression (podoplanin and prosurfactant protein-C) than rats administered BM-Muse cells. Rats receiving preterm-UC-Muse cells showed statistically superior results to those receiving term-UC-Muse cells in many of the measures. These findings are thought to be due to higher expression of genes related to cell migration, lung differentiation, and cell adhesion.ConclusionPreterm UC-Muse cells deliver more efficient therapeutic effects than term UC- and BM-Muse cells for treating BLM-induced lung injury in a rat model.

Lipocalin-2 as a prognostic marker in patients with acute exacerbation of idiopathic pulmonary fibrosis

BackgroundLipocalin-2 (LCN2) is a secretory glycoprotein upregulated by oxidative stress; moreover, patients with idiopathic pulmonary fibrosis (IPF) have shown increased LCN2 levels in bronchoalveolar lavage fluid (BALF). This study aimed to determine whether circulatory LCN2 could be a systemic biomarker in patients with IPF and to investigate the role of LCN2 in a bleomycin-induced lung injury mouse model.MethodsWe measured serum LCN2 levels in 99 patients with stable IPF, 27 patients with acute exacerbation (AE) of IPF, 51 patients with chronic hypersensitivity pneumonitis, and 67 healthy controls. Further, LCN2 expression in lung tissue was evaluated in a bleomycin-induced lung injury mouse model, and the role of LCN2 was investigated using LCN2-knockout (LCN2 -/-) mice.ResultsSerum levels of LCN2 were significantly higher in patients with AE-IPF than in the other groups. The multivariate Cox proportional hazards model showed that elevated serum LCN2 level was an independent predictor of poor survival in patients with AE-IPF. In the bleomycin-induced lung injury mouse model, a higher dose of bleomycin resulted in higher LCN2 levels and shorter survival. Bleomycin-treated LCN2 -/- mice exhibited increased BALF cell and protein levels as well as hydroxyproline content. Moreover, compared with wild-type mice, LCN2-/- mice showed higher levels of circulatory 8-isoprostane as well as lower Nrf-2, GCLC, and NQO1 expression levels in lung tissue following bleomycin administration.ConclusionsOur findings demonstrate that serum LCN2 might be a potential prognostic marker of AE-IPF. Moreover, LCN2 expression levels may reflect the severity of lung injury, and LCN2 may be a protective factor against bleomycin-induced acute lung injury and oxidative stress.

Development of novel technology for the visualization and quantitation of microvascular progenitor-driven adaptive angiogenesis during chronic lung disease

Introduction: Adaptive angiogenesis can drive repair or underlie the pathogenesis of tissue remodeling. Pulmonary vascular dysfunction is a major manifestation of chronic lung disease (CLD) but the role of angiogenesis in CLD is not well defined. Microvascular capillaries are the vessels most affected by pruning and remodeling in the lung resulting in reduced capillary length and density. Our lab has previously demonstrated that microvascular progenitors (ABCG2+ MVPCs) drive adaptive angiogenesis or loss of tissue structure downstream of the Wnt or mTOR signaling pathways. Therefore, we hypothesize that visualization of the pulmonary microvasculature and MVPC-driven adaptive angiogenesis in three-dimensions is essential to define the role of MVPC-driven adaptive angiogenesis during repair or the pathogenesis of CLD. Methods: To evaluate the contribution of Wnt activated MVPCs to angiogenesis in three-dimensions during injury repair, we crossed ABCG2-CreERT2 mice to a BOE strain (flΔex3) with stabilized β-catenin expression and a fluorescent membrane eGFP reporter strain to facilitate lineage tracing. The development and resolution of fibrosis were induced in vivo via aspiration of bleomycin sulfate (0.05U). Mice were retro-orbitally perfused with fluorescently conjugated tomato lectin. Lung tissue was inflated with agarose and up to 500μm thickness precision cut lung slices (PCLS) were mounted for imaging. PCLS were imaged on the Zeiss LSM900 and Z-stacks were obtained. Imaris software (Oxford Instruments) was used for three-dimensional reconstruction and FIJI and Wimasis Image Analysis (Onimagin Technologies) were utilized to segment vessels, quantitate vessel density, volume, diameter, and length. Results: Following perfusion, vascular staining of both the lung and kidney was observed, suggesting successful systemic perfusion and labeling. The pulmonary microvasculature was visualized to a diameter of 1-3μm in high fidelity. We observed a significant reduction in vessel density in mice treated with bleomycin compared to mice treated with a vehicle control. In our three-dimensional reconstructions we observed increased density of ABCG2+ MVPC-derived de novo angiogenic structures in areas of fibrosis. Conclusion: De novo angiogenesis driven by ABCG2+ MVPCs likely plays a role in tissue repair following bleomycin induced lung injury. In addition, we have developed a simple and reliable perfusion technique allowing for the visualization and quantitation of the pulmonary microvasculature in three-dimensions. This tool will allow the quantitation of microvessel remodeling underlying pathogenesis and repair in multiple lung injury models. Thus, this methodology will further support the study and understanding of angiogenesis during CLD and repair following injury. This approach may be utilized in various organ systems to visualize microvasculature structure and its role in disease. NIA RAG073317 NHLBI 1R35HL161238. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex differences in Acute Lung Injury (ALI)-Induced Respiratory Functional Changes in a Bleomycin Rat Model

Introduction: Acute lung injury (ALI) is a pathology characterized primarily by profound disruption of the epithelial-endothelial interface, leukocyte migration into the alveolar space, leading to disordered gas exchange and consequential respiratory failure. There exist several animal models to study the pathophysiology of ALI. Bleomycin, a cancer therapeutic, has been shown to consistently induce the ALI phenotype in rodent models. We have previously shown that intra-tracheal bleomycin instillation in rats produces deleterious changes in respiratory function in both acute and recovery phases of ALI. In this study, we sought to elucidate the effect of sex bleomycin-induced ALI. Methods: To do this, cohorts of male and female Sprague-Dawley rats were measured using whole-body plethysmography (WBP) where the following respiratory parameters were recorded prior to bleomycin exposure (Week 0): Volume of Oxygen consumed normalized to body weight (VO2/kg), Respiratory rate (RR), Tidal Volume normalized to body weight (TV/kg), Minute Ventilation normalized to body weight (MV/kg), enhanced pause (Penh), pause (PAU), ratio rate of achieving peak expiatory flow (Rpef), peak inspiratory flow (box) (PIFb), peak expiratory flow box (PEFb), inspiratory time (Ti), expiratory time (Te), expiratory flow at the point of 50%TV (EF50), end-inspiratory pause (EIP), end-expiratory pause (EEP), relaxation time (Tr) and time of brake (TB). A single dose of intra-tracheal bleomycin (2.5mg/kg) was instilled and subsequent WBP recordings were performed weekly during the acute phase of ALI (Week 1), and the recovery phase of ALI (Week 4). Results: Our results show that bleomycin-induced ALI resulted in greater DTE (Weeks 1-3) and DTR (Weeks 1-2) in females compared to males. Measures of airway obstruction were also shown to be sex-dependent, as DRpef (Week 1), DPAU (Weeks 2-4), and DPenh (Weeks 2-4) were significantly different between males and females. EF50, a metric highly correlated with lung pathologies, remained unchanged between sexes post-ALI. While inducing ALI did not alter DPIFb between the sexes, DPEFb was elevated among males compared to females (Weeks 2,4). DEIP and DEEP both showed sex-dependent changes post-ALI, with females displaying a greater decrease compared to males. Additionally, there was a greater decrease in DTV/kg among females compared to males post-ALI (Weeks 1-3). DRR, DMV/kg, and DVO2/kg were unchanged across the 4 weeks post-ALI between sexes. Conclusion: Our study reveals sex-specific nuances in bleomycin-induced acute lung injury (ALI) in Sprague-Dawley rats. Differential responses in airway obstruction measures and distinct impacts on respiratory dynamics underscore the importance of considering sex in researching ALI. This work is supported by NIH R01HL152160-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Bleomycin induces senescence and repression of DNA repair via downregulation of Rad51

BackgroundBleomycin, a potent antitumor agent, is limited in clinical use due to the potential for fatal pulmonary toxicity. The accelerated DNA damage and senescence in alveolar epithelial cells (AECs) is considered a key factor in the development of lung pathology. Understanding the mechanisms for bleomycin-induced lung injury is crucial for mitigating its adverse effects.MethodsHuman lung epithelial (A549) cells were exposed to bleomycin and subsequently assessed for cellular senescence, DNA damage, and double-strand break (DSB) repair. The impact of Rad51 overexpression on DSB repair and senescence in AECs was evaluated in vitro. Additionally, bleomycin was intratracheally administered in C57BL/6 mice to establish a pulmonary fibrosis model.ResultsBleomycin exposure induced dose- and time-dependent accumulation of senescence hallmarks and DNA lesions in AECs. These effects are probably due to the inhibition of Rad51 expression, consequently suppressing homologous recombination (HR) repair. Mechanistic studies revealed that bleomycin-mediated transcriptional inhibition of Rad51 might primarily result from E2F1 depletion. Furthermore, the genetic supplement of Rad51 substantially mitigated bleomycin-mediated effects on DSB repair and senescence in AECs. Notably, decreased Rad51 expression was also observed in the bleomycin‐induced mouse pulmonary fibrosis model.ConclusionsOur works suggest that the inhibition of Rad51 plays a pivotal role in bleomycin-induced AECs senescence and lung injury, offering potential strategies to alleviate the pulmonary toxicity of bleomycin.

Microscopic computed tomography with AI-CNN-powered image analysis: the path to phenotype bleomycin-induced lung injury.

Bleomycin (BLM)-induced lung injury in mice is a valuable model for investigating the molecular mechanisms that drive inflammation and fibrosis and for evaluating potential therapeutic approaches to treat the disease. Given high variability in the BLM model, it is critical to accurately phenotype the animals in the course of an experiment. In the present study, we aimed to demonstrate the utility of microscopic computed tomography (µCT) imaging combined with an artificial intelligence (AI)-convolutional neural network (CNN)-powered lung segmentation for rapid phenotyping of BLM mice. µCT was performed in freely breathing C57BL/6J mice under isoflurane anesthesia on days 7 and 21 after BLM administration. Terminal invasive lung function measurement and histological assessment of the left lung collagen content were conducted as well. µCT image analysis demonstrated gradual and time-dependent development of lung injury as evident by alterations in the lung density, air-to-tissue volume ratio, and lung aeration in mice treated with BLM. The right and left lung were unequally affected. µCT-derived parameters such as lung density, air-to-tissue volume ratio, and nonaerated lung volume correlated well with the invasive lung function measurement and left lung collagen content. Our study demonstrates the utility of AI-CNN-powered µCT image analysis for rapid and accurate phenotyping of BLM mice in the course of disease development and progression.NEW & NOTEWORTHY Microscopic computed tomography (µCT) imaging combined with an artificial intelligence (AI)-convolutional neural network (CNN)-powered lung segmentation is a rapid and powerful tool for noninvasive phenotyping of bleomycin mice over the course of the disease. This, in turn, allows earlier and more reliable identification of therapeutic effects of new drug candidates, ultimately leading to the reduction of unnecessary procedures in animals in pharmacological research.

Evaluation of interleukin-1 and interleukin-6 receptor antagonists in a murine model of acute lung injury.

The acute exudative phase of acute respiratory distress syndrome (ARDS), a severe form of respiratory failure, is characterized by alveolar damage, pulmonary oedema, and an exacerbated inflammatory response. There is no effective treatment for this condition, but based on the major contribution of inflammation, anti-inflammatory strategies have been evaluated in animal models and clinical trials, with conflicting results. In COVID-19 ARDS patients, interleukin (IL)-1 and IL-6 receptor antagonists (IL-1Ra and IL-6Ra, kineret and tocilizumab, respectively) have shown some efficacy. Moreover, we have previously developed novel peptides modulating IL-1R and IL-6R activity (rytvela and HSJ633, respectively) while preserving immune vigilance and cytoprotective pathways. We aimed to assess the efficacy of these novel IL-1Ra and IL-6Ra, compared to commercially available drugs (kineret, tocilizumab) during the exudative phase (day 7) of bleomycin-induced acute lung injury (ALI) in mice. Our results first showed that none of the IL-1Ra and IL-6Ra compounds attenuated bleomycin-induced weight loss and venous increase. Histological analyses and lung water content measurements also showed that these drugs did not improve lung injury scores or pulmonary oedema, after the bleomycin challenge. Finally, IL-1Ra and IL-6Ra failed to alleviate the inflammatory status of the mice, as indicated by cytokine levels and alveolar neutrophil infiltration. Altogether, these results indicate a lack of beneficial effects of IL-1R and IL-6R antagonists on key parameters of ALI in the bleomycin mouse model.

Antrodia cinnamomea extract alleviates bleomycin-induced pulmonary fibrosis in mice by inhibiting the mTOR pathway

BackgroundPulmonary fibrosis is a progressive diffuse parenchymal lung disorder with a high mortality rate. Studies have indicated that injured lung tissues release various pro-inflammatory factors, and produce a large amount of nitric oxide. There is also accumulation of collagen and oxidative stress-induced injury, collectively leading to pulmonary fibrosis. Antrodia cinnamomea is an endemic fungal growth in Taiwan, and its fermented extracts exert anti-inflammatory effects to alleviate liver damages. Hence, we hypothesized and tested the feasibility of using A. cinnamomea extracts for treatment of pulmonary fibrosis. MethodsThe TGF-β1-induced human lung fibroblast cells (MRC-5) in vitro cell assay were used to evaluate the effects of A. cinnamomea extracts on the collagen production in MRC-5. Eight-week-old ICR mice were intratracheally administered bleomycin and then fed with an A. cinnamomea extract on day 3 post-administration of bleomycin. At day 21 post-bleomycin administration, the pulmonary functional test, the expression level of inflammation- and fibrosis-related genes in the lung tissue, and the histopathological change were examined. ResultsThe A. cinnamomea extract significantly attenuated the expression level of collagen in the TGF-β1-induced MRC-5 cells. In the A. cinnamome-treated bleomycin-induced lung fibrotic mice, the bodyweight increased, pulmonary functions improved, the lung tissues expression level of inflammatory factor and the fibrotic indicator were decreased, and the histopathological results showed the reduction of thickening of the inter-alveolar septa. ConclusionsThe Antrodia cinnamomea extract significant protects mice against bleomycin-induced lung injuries through improvement of body weight gain and lung functions, and attenuation of expression of inflammatory and fibrotic indicators.

SPAUTIN-1 alleviates LPS-induced acute lung injury by inhibiting NF-κB pathway in neutrophils

BackgroundAcute lung injury (ALI) is an inflammatory condition characterized by acute damage to lung tissue. SPAUTIN-1, recognized as a small molecule drug targeting autophagy and USP10/13, has been reported for its potential to inhibit oxidative stress damage in various tissue injuries. However, the role and mechanism of SPAUTIN-1 in ALI remain unclear. This study aims to elucidate the protective effects of SPAUTIN-1 on ALI, with a particular focus on its role and mechanism in pulmonary inflammatory responses. MethodsLipopolysaccharides (LPS) were employed to induce inflammation-mediated ALI. Bleomycin was used to induce non-inflammation-mediated ALI. The mechanism of SPAUTIN-1 action was identified through RNA-Sequencing and subsequently validated in mouse primary cells. Tert-butyl hydroperoxide (TBHP) was utilized to create an in vitro model of lung epithelial cell oxidative stress with MLE-12 cells. ResultsSPAUTIN-1 significantly mitigated LPS-induced lung injury and inflammatory responses, attenuated necroptosis and apoptosis in lung epithelial cells, and inhibited autophagy in leukocytes and epithelial cells. However, SPAUTIN-1 exhibited no significant effect on bleomycin-induced lung injury. RNA-sequencing results demonstrated that SPAUTIN-1 significantly inhibited the NF-κB signaling pathway in leukocytes, a finding consistently confirmed by mouse primary cell assays. In vitro experiments further revealed that SPAUTIN-1 effectively mitigated oxidative stress injury in MLE-12 cells induced by TBHP. ConclusionSPAUTIN-1 alleviated LPS-induced inflammatory injury by inhibiting the NF-κB pathway in leukocytes and protected epithelial cells from oxidative damage, positioning it as a potential therapeutic candidate for ALI.

Capsaicin ameliorate pulmonary fibrosis via antioxidant Nrf-2/ PPAR- γ pathway activation and inflammatory TGF-β1/ NF-κB/COX II pathway inhibition.

Bleomycin is an effective antibiotic with a significant anticancer properties, but its use is limited due to its potential to induce dose-dependent pulmonary fibrosis. Therefore, this study aimed to assess the therapeutic potential of Capsaicin as an additional treatment to enhance patient tolerance to Bleomycin compared to the antifibrotic drug Pirfenidone. Pulmonary fibrosis was induced in rats through by a single intratracheal Bleomycin administration in day zero, followed by either Capsaicin or Pirfenidone treatment for 7days. After the animals were sacrificed, their lungs were dissected and examined using various stains for macroscopic and histopathological evaluation. Additionally, the study assessed various antioxidant, anti-inflammatory, and antifibrotic parameters were assessed. Rats exposed to Bleomycin exhibited visible signs of fibrosis, histopathological alterations, increased collagen deposition, and elevated mucin content. Bleomycin also led to heightened increased inflammatory cells infiltration in the bronchoalveolar lavage, elevated fibrosis biomarkers such as hydroxyproline, alpha-smooth muscle actin (α-SMA) and transforming growth factor-beta (TGF-β1), increased inflammatory markers including tumor necrosis factor-alpha (TNF-α), interlukine-6 (Il-6), interlukine-1β (Il-1β) nuclear factor-kappa B (NF-κB), and Cyclooxygenase-2 (COX-2), and transforming growth factor-beta (TGF-β1),. Furthermore, it reduced the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ), increased oxidative stress biomarkers like nitric oxide (NO), malondialdehyde (MDA), myeloperoxidase (MPO) and protein carbonyl. Bleomycin also decreased the expression of nuclear factor erythroid 2-related factor 2 (Nrf-2), reduced glutathione (GSH), total antioxidant capacity, and the activities of catalase and superoxide dismutase (SOD). Treating the animals with Capsaicin and Pirfenidone following Bleomycin exposure resulted in improved lung macroscopic and microscopic characteristics, reduced collagen deposition (collagen I and collagen III) and mucin content, decreased inflammatory cell infiltration, lowered levels of hydroxyproline, α-SMA, and TGF-β1, decreased TNF-α, Il-6, Il-1β, NF-κB, and COX-2, increased PPAR-γ and Nrf-2 expression, and improvement improved in all oxidative stress biomarkers. In summary, Capsaicin demonstrates significant antifibrotic activity against Bleomycin-induced lung injury that may be attributed, at least in part, to the antioxidant and anti-inflammatory activities of Capsaicin mediated by upregulation of PPAR-γ and Nrf-2 expression and decreasing. TGF-β1, NF-κB and COX II proteins concentrations.

Function of KvLQT1 potassium channels in a mouse model of bleomycin-induced acute lung injury.

Acute respiratory distress syndrome (ARDS) is characterized by an exacerbated inflammatory response, severe damage to the alveolar-capillary barrier and a secondary infiltration of protein-rich fluid into the airspaces, ultimately leading to respiratory failure. Resolution of ARDS depends on the ability of the alveolar epithelium to reabsorb lung fluid through active transepithelial ion transport, to control the inflammatory response, and to restore a cohesive and functional epithelium through effective repair processes. Interestingly, several lines of evidence have demonstrated the important role of potassium (K+) channels in the regulation of epithelial repair processes. Furthermore, these channels have previously been shown to be involved in sodium/fluid absorption across alveolar epithelial cells, and we have recently demonstrated the contribution of KvLQT1 channels to the resolution of thiourea-induced pulmonary edema in vivo. The aim of our study was to investigate the role of the KCNQ1 pore-forming subunit of KvLQT1 channels in the outcome of ARDS parameters in a model of acute lung injury (ALI). We used a molecular approach with KvLQT1-KO mice challenged with bleomycin, a well-established ALI model that mimics the key features of the exudative phase of ARDS on day 7. Our data showed that KvLQT1 deletion exacerbated the negative outcome of bleomycin on lung function (resistance, elastance and compliance). An alteration in the profile of infiltrating immune cells was also observed in KvLQT1-KO mice while histological analysis showed less interstitial and/or alveolar inflammatory response induced by bleomycin in KvLQT1-KO mice. Finally, a reduced repair rate of KvLQT1-KO alveolar cells after injury was observed. This work highlights the complex contribution of KvLQT1 in the development and resolution of ARDS parameters in a model of ALI.

Gene therapy with AAV9-SGPL1 in an animal model of lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung that leads rapidly to respiratory failure. Novel approaches to treatment are urgently needed. The bioactive lipid sphingosine-1-phosphate (S1P) is increased in IPF lungs and promotes proinflammatory and profibrotic TGF-β signaling. Hence, decreasing lung S1P represents a potential therapeutic strategy for IPF. S1P is degraded by the intracellular enzyme S1P lyase (SPL). Here we find that a knock-in mouse with a missense SPL mutation mimicking human disease resulted in reduced SPL activity, increased S1P, increased TGF-β signaling, increased lung fibrosis, and higher mortality after injury compared to wild type (WT). We then tested adeno-associated virus 9 (AAV9)-mediated overexpression of human SGPL1 (AAV-SPL) in mice as a therapeutic modality. Intravenous treatment with AAV-SPL augmented lung SPL activity, attenuated S1P levels within the lungs, and decreased injury-induced fibrosis compared to controls treated with saline or only AAV. We confirmed that AAV-SPL treatment led to higher expression of SPL in the epithelial and fibroblast compartments during bleomycin-induced lung injury. Additionally, AAV-SPL decreased expression of the profibrotic cytokines TNFα and IL1β as well as markers of fibroblast activation, such as fibronectin (Fn1), Tgfb1, Acta2, and collagen genes in the lung. Taken together, our results provide proof of concept for the use of AAV-SPL as a therapeutic strategy for the treatment of IPF. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Bleomycin-induced modulations of PARP 1 activity, NAD+ and PARG content in rat lung nuclei

Bleomycin-induced lung pathology in rodents is a well recognized animal model widely used for evaluation of new therapeutic approaches in treatment of lung inflammation and fibrotic diseases. It is documented that poly(ADP-ribose)polymerase 1 activity has a significant role in development of inflammatory processes in the heart, liver and brain. Herein, we used biochemical and immunochemical methods for estimation of poly(ADP-ribose)polymerase 1 (PARP 1) activity, NAD+ and poly(ADP-ribose)glycohydrolase (PARG) protein content in rat lung nuclei during the inflammatory phase in a bleomycin-induced one-hit rat model. To evaluate the influence of bleomycin – induced alterations in DNA structure on regulation of poly(ADP-ribose)polymerase 1 activation pathways, we isolated DNA from nuclei of lung tissues in the phase of acute lung inflammation induced by bleomycin, and DNA melting profiles were investigated. In the present study we investigated whether naturally occurring water-soluble polyphenol tannic acid with widely accepted anti-fibrotic and anti-inflammatory effects can influence poly(ADP-ribose)polymerase 1 activity, NAD+ and poly(ADP-ribose)glycohydrolase protein content in nuclear fraction isolated from rat lung tissues in a bleomycin-induced acute lung injury model. It was demonstrated that NAD+ level and poly(ADP-ribose)glycohydrolase protein content decreased in rat lung nuclei during the inflammatory phase in the bleomycin-induced acute lung injury model. Treatment of rats with tannic acid enhanced the effects displayed by bleomycin in lung nuclei, thus indicating synergistic interaction with the drug in the field covering PARP 1 activity, poly(ADP-ribose)glycohydrolase (PARG) protein and NAD+ content in lung nuclei. We observed PARP 1 inhibition in nuclei of lung tissue during the inflammatory phase in the bleomycin-induced acute lung injury rat model, which could be coupled with the drop of NAD+ level in nuclei. In the present study we highlighted that bleomycin (BLM) can induce DNA destabilization in lung nuclei. It was proposed that bleomycin-induced modulations in DNA structure could hamper PARP 1 binding with DNA and down-regulate the enzyme activating pathway in lung nuclei. The role of poly(ADP-ribose)glycohydrolase depletion in lung nuclei and sequential accumulation of poly(ADP-ribose)polymers in lung cells, which triggers their destruction and tissues damage, was proposed. It is suggested that in the light of synergistic interaction between bleomycin and tannic acid (TA) the anti-inflammatory role of tannic acid should be repurposed.

Inhibition of sphingosine 1-phosphate receptor 3 ameliorates bleomycin-induced pulmonary fibrosis by suppressing macrophage M2 polarization

Pulmonary fibrosis is a devastating lung disease without effective treatment options. Sphingosine-1-phosphate receptor 3 (S1pr3), a receptor for the lipid signaling molecule sphingosine-1-phosphate, has been shown to mediate the development of pulmonary fibrosis, although the underlying mechanism is not fully understood. Here, we found increased expression of S1pr3 in the lung during the process of bleomycin-induced pulmonary fibrosis in mice and specific overexpression of S1pr3 in the infiltrated M2 macrophages. We constructed LysM-Cre+/S1pr3flox/flox mice, in which S1pr3 was conditionally depleted in myeloid cells, and this depletion protected mice from bleomycin-induced lung injury and fibrosis, with reduced M2 macrophage accumulation in the lung. Increased S1pr3 expression was found in bone marrow-derived macrophages after alternatively activated by IL4 ex vivo, while loss of S1pr3 attenuated IL-4-induced M2 polarization in bone marrow-derived macrophages by repressing the PI3K/Akt-Stat3 signaling pathway. Moreover, the S1pr3 inhibitors CAY10444 and TY52156 exerted protective effects on pulmonary fibrosis in mice. Taken together, our research showed that inhibition of S1pr3 ameliorates bleomycin-induced pulmonary fibrosis by reducing macrophage M2 polarization via the PI3K/Akt-Stat3 signaling pathway, indicating that S1pr3 may be a potential target for pulmonary fibrosis treatment.