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Abstract This study aimed to investigate the potential anti-fibrotic activity of vinpocetine in an experimental model of pulmonary fibrosis by bleomycin and in the MRC-5 cell line. Pulmonary fibrosis was induced in BALB/c mice by oropharyngeal aspiration of a single dose of bleomycin (5 mg/kg). The remaining induced animals received a daily dose of pirfenidone (as a standard anti-fibrotic drug) (300 mg/kg/PO) and vinpocetine (20 mg/kg/PO) on day 7 of the induction till the end of the experiment (day 21). The results of the experiment revealed that vinpocetine managed to alleviate the fibrotic endpoints by statistically improving (P ≤ 0.05) the weight index, histopathological score, reduced expression of fibrotic-related proteins in immune-stained lung sections, as well as fibrotic markers measured in serum samples. It also alleviated tissue levels of oxidative stress and inflammatory and pro-fibrotic mediators significantly elevated in bleomycin-only induced animals (P ≤ 0.05). Vinpocetine managed to express a remarkable attenuating effect in pulmonary fibrosis both in vivo and in vitro either directly by interfering with the classical TGF-β1/Smad2/3 signaling pathway or indirectly by upregulating the expression of Nrf2 enhancing the antioxidant system, activating PPAR-γ and downregulating the NLRP3/NF-κB pathway making it a candidate for further clinical investigation in cases of pulmonary fibrosis.

Background: Pulmonary 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. Methods: The 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. Results: The 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. Conclusions: The 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.

The bleomycin-induced pulmonary fibrosis mouse model is commonly used in idiopathic pulmonary fibrosis research, but its cellular and molecular changes and efficiency as a model at the molecular level are not fully understood. In this study, we used spatial transcriptome technology to investigate the cellular and molecular changes in the lungs of bleomycin-induced pulmonary fibrosis mouse models. Our analyses revealed cell dynamics during fibrosis in epithelial cells, mesenchymal cells, immunocytes, and erythrocytes with their spatial distribution available. We confirmed the differentiation of the alveolar type II (AT2) cell type expressing Krt8, and we inferred their trajectories from both the AT2 cells and club cells. In addition to the fibrosis process, we also noticed evidence of self-resolving, especially to identify possible self-resolving related genes, including Prkca. Our findings provide insights into the cellular and molecular mechanisms underlying fibrosis resolution and represent the first spatiotemporal transcriptome dataset of the bleomycin-induced fibrosis mouse model.

Abstract Background Interstitial lung diseases (ILDs) can be induced and even exacerbated by radiotherapy in thoracic cancer patients. The roles of immune responses underlying the development of these severe lung injuries are still obscure and need to be investigated. Methods A severe lung damage murine model was established by delivering 16 Gy X-rays to the chest of mice that had been pre-treated with bleomycin (BLM) and thus hold ILDs. Bioinformatic analyses were performed on the GEO datasets of radiation-induced lung injury (RILI) and BLM-induced pulmonary fibrosis (BIPF), and RNA-sequencing data of the severely damaged lung tissues. The screened differentially expressed genes (DEGs) were verified in lung epithelial cell lines by qRT-PCR assay. The injured lung tissue pathology was analyzed with H&E and Masson’s staining, and immunohistochemistry staining. The macrophage chemotaxis and activity promoted by the stressed epithelial cells were determined by using a cell co-culture system. The expressions of p21 in MLE-12 and Beas-2B cells were detected by qRT-PCR, western blot, and immunofluorescence. The concentration of CCL7 in cell supernatant was measured by ELISA assay. In some experiments, Beas-2B cells were transfected with p21-siRNA or CCL7-siRNA before irradiation and/or BLM treatment. Results After the treatment of irradiation and/or BLM, the inflammatory and immune responses, chemokine-mediated signaling pathways were steadily activated in the severely injured lung, and p21 was screened out by the bioinformatic analysis and further verified to be upregulated in both mouse and human lung epithelial cell lines. The expression of P21 was positively correlated with macrophage infiltration in the injured lung tissues. Co-culturing with stressed Beas-2B cells or its conditioned medium containing CCL7 protein, U937 macrophages were actively polarized to M1-phase and their migration ability was obviously increased along with the damage degree of Beas-2B cells. Furthermore, knockdown p21 reduced CCL7 expression in Beas-2B cells and then decreased the chemotaxis of co-cultured macrophages. Conclusions P21 promoted CCL7 release from the severely injured lung epithelial cell lines and contributed to the macrophage chemotaxis in vitro, which provides new insights for better understanding the inflammatory responses in lung injury.

Abstract Background Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease (ILD) with limited treatment options. Interleukin-33 (IL-33) is proposed to play a role in the development of IPF however the exclusive use of prophylactic dosing regimens means that the therapeutic benefit of targeting this cytokine in IPF is unclear. Methods IL-33 expression was assessed in ILD lung sections and human lung fibroblasts (HLFs) by immunohistochemistry and gene/protein expression and responses of HLFs to IL-33 stimulation measured by qPCR. In vivo, the fibrotic potential of IL-33:ST2 signalling was assessed using a murine model of bleomycin (BLM)-induced pulmonary fibrosis and therapeutic dosing with an ST2-Fc fusion protein. Lung and bronchoalveolar lavage fluid were collected for measurement of inflammatory and fibrotic endpoints. Human precision-cut lung slices (PCLS) were stimulated with transforming growth factor-β (TGFβ) or IL-33 and fibrotic readouts assessed. Results IL-33 was expressed by fibrotic fibroblasts in situ and was increased by TGFβ treatment in vitro. IL-33 treatment of HLFs did not induce IL6, CXCL8, ACTA2 and COL1A1 mRNA expression with these cells found to lack the IL-33 receptor ST2. Similarly, IL-33 stimulation had no effect on ACTA2, COL1A1, FN1 and fibronectin expression by PCLS. Despite having effects on inflammation suggestive of target engagement, therapeutic dosing with the ST2-Fc fusion protein failed to reduce BLM-induced fibrosis measured by hydroxyproline content or Ashcroft score. Conclusions Together these findings suggest the IL-33:ST2 axis does not play a central fibrogenic role in the lungs with therapeutic blockade of this pathway unlikely to surpass the current standard of care for IPF.

Abstract Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease characterized by alveolar epithelial cell injury and lung fibroblast overactivation. At present, only two drugs are approved by the FDA for the treatment of IPF, including the synthetic pyridinone drug, pirfenidone, and the tyrosine kinase inhibitor, nintedanib. Avitinib (AVB) is a novel oral and potent third-generation tyrosine kinase inhibitor for treating non-small cell lung cancer (NSCLC). However, the role of avitinib in pulmonary fibrosis has not yet been established. In the present study, we used in vivo and in vitro models to evaluate the role of avitinib in pulmonary fibrosis. In vivo experiments first verified that avitinib significantly alleviated bleomycin-induced pulmonary fibrosis in mice. Further in vitro molecular studies indicated that avitinib inhibited myofibroblast activation, migration and extracellular matrix (ECM) production in NIH-3T3 cells, mainly by inhibiting the TGF-β1/Smad3 signalling pathways. The cellular experiments also indicated that avitinib improved alveolar epithelial cell injury in A549 cells. In conclusion, the present findings demonstrated that avitinib attenuates bleomycin-induced pulmonary fibrosis in mice by inhibiting alveolar epithelial cell injury and myofibroblast activation.

Idiopathic pulmonary fibrosis (IPF) is an irreversible and life-threatening lung disease of unknown etiology presenting only a few treatment options. TGF-β signaling orchestrates a cascade of events driving pulmonary fibrosis (PF). Notably, recent research has affirmed the augmentation of TGF-β receptor (TβR) signaling via HSP90 activation. HSP90, a molecular chaperone, adeptly stabilizes and folds TβRs, thus intricately regulating TGF-β1 signaling. Our investigation illuminated the impact of alvespimycin, an HSP90 inhibitor, on TGF-β-mediated transcriptional responses by inducing destabilization of TβRs. This outcome stems from the explicit interaction of TβR subtypes I and II with HSP90, where they are clients of this cellular chaperone. It is worth noting that regulation of proteasome-dependent degradation of TβRs is a critical standpoint in the termination of TGF-β signal transduction. Oleuropein, the principal bioactive compound found in Olea europaea, is acknowledged for its role as a proteasome activator. In this study, our aim was to explore the efficacy of a combined therapy involving oleuropein and alvespimycin for the treatment of PF. We employed a PF rat model that was induced by intratracheal bleomycin infusion. The application of this dual therapy yielded a noteworthy impediment to the undesired activation of TGF-β/mothers against decapentaplegic homologs 2 and 3 (SMAD2/3) signaling. Consequently, this novel combination showcased improvements in both lung tissue structure and function while also effectively restraining key fibrosis markers such as PDGF-BB, TIMP-1, ACTA2, col1a1, and hydroxyproline. On a mechanistic level, our findings unveiled that the antifibrotic impact of this combination therapy likely stemmed from the enhanced degradation of both TβRI and TβRII. In conclusion, the utilization of proteasomal activators in conjunction with HSP90 inhibitors ushers in a promising frontier for the management of PF. [ABSTRACT FROM AUTHOR]

Background: Interstitial lung diseases (ILDs) can be induced and even exacerbated by radiotherapy in thoracic cancer patients. The roles of immune responses underlying the development of these severe lung injuries are still obscure and need to be investigated. Methods: A severe lung damage murine model was established by delivering 16 Gy X-rays to the chest of mice that had been pre-treated with bleomycin (BLM) and thus hold ILDs. Bioinformatic analyses were performed on the GEO datasets of radiation-induced lung injury (RILI) and BLM-induced pulmonary fibrosis (BIPF), and RNA-sequencing data of the severely damaged lung tissues. The screened differentially expressed genes (DEGs) were verified in lung epithelial cell lines by qRT-PCR assay. The injured lung tissue pathology was analyzed with H&E and Masson's staining, and immunohistochemistry staining. The macrophage chemotaxis and activity promoted by the stressed epithelial cells were determined by using a cell co-culture system. The expressions of p21 in MLE-12 and Beas-2B cells were detected by qRT-PCR, western blot, and immunofluorescence. The concentration of CCL7 in cell supernatant was measured by ELISA assay. In some experiments, Beas-2B cells were transfected with p21-siRNA or CCL7-siRNA before irradiation and/or BLM treatment. Results: After the treatment of irradiation and/or BLM, the inflammatory and immune responses, chemokine-mediated signaling pathways were steadily activated in the severely injured lung, and p21 was screened out by the bioinformatic analysis and further verified to be upregulated in both mouse and human lung epithelial cell lines. The expression of P21 was positively correlated with macrophage infiltration in the injured lung tissues. Co-culturing with stressed Beas-2B cells or its conditioned medium containing CCL7 protein, U937 macrophages were actively polarized to M1-phase and their migration ability was obviously increased along with the damage degree of Beas-2B cells. Furthermore, knockdown p21 reduced CCL7 expression in Beas-2B cells and then decreased the chemotaxis of co-cultured macrophages. Conclusions: P21 promoted CCL7 release from the severely injured lung epithelial cell lines and contributed to the macrophage chemotaxis in vitro, which provides new insights for better understanding the inflammatory responses in lung injury. [ABSTRACT FROM AUTHOR]

Background: Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease (ILD) with limited treatment options. Interleukin-33 (IL-33) is proposed to play a role in the development of IPF however the exclusive use of prophylactic dosing regimens means that the therapeutic benefit of targeting this cytokine in IPF is unclear. Methods: IL-33 expression was assessed in ILD lung sections and human lung fibroblasts (HLFs) by immunohistochemistry and gene/protein expression and responses of HLFs to IL-33 stimulation measured by qPCR. In vivo, the fibrotic potential of IL-33:ST2 signalling was assessed using a murine model of bleomycin (BLM)-induced pulmonary fibrosis and therapeutic dosing with an ST2-Fc fusion protein. Lung and bronchoalveolar lavage fluid were collected for measurement of inflammatory and fibrotic endpoints. Human precision-cut lung slices (PCLS) were stimulated with transforming growth factor-β (TGFβ) or IL-33 and fibrotic readouts assessed. Results: IL-33 was expressed by fibrotic fibroblasts in situ and was increased by TGFβ treatment in vitro. IL-33 treatment of HLFs did not induce IL6, CXCL8, ACTA2 and COL1A1 mRNA expression with these cells found to lack the IL-33 receptor ST2. Similarly, IL-33 stimulation had no effect on ACTA2, COL1A1, FN1 and fibronectin expression by PCLS. Despite having effects on inflammation suggestive of target engagement, therapeutic dosing with the ST2-Fc fusion protein failed to reduce BLM-induced fibrosis measured by hydroxyproline content or Ashcroft score. Conclusions: Together these findings suggest the IL-33:ST2 axis does not play a central fibrogenic role in the lungs with therapeutic blockade of this pathway unlikely to surpass the current standard of care for IPF. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease characterized by alveolar epithelial cell injury and lung fibroblast overactivation. At present, only two drugs are approved by the FDA for the treatment of IPF, including the synthetic pyridinone drug, pirfenidone, and the tyrosine kinase inhibitor, nintedanib. Avitinib (AVB) is a novel oral and potent third-generation tyrosine kinase inhibitor for treating non-small cell lung cancer (NSCLC). However, the role of avitinib in pulmonary fibrosis has not yet been established. In the present study, we used in vivo and in vitro models to evaluate the role of avitinib in pulmonary fibrosis. In vivo experiments first verified that avitinib significantly alleviated bleomycin-induced pulmonary fibrosis in mice. Further in vitro molecular studies indicated that avitinib inhibited myofibroblast activation, migration and extracellular matrix (ECM) production in NIH-3T3 cells, mainly by inhibiting the TGF-β1/Smad3 signalling pathways. The cellular experiments also indicated that avitinib improved alveolar epithelial cell injury in A549 cells. In conclusion, the present findings demonstrated that avitinib attenuates bleomycin-induced pulmonary fibrosis in mice by inhibiting alveolar epithelial cell injury and myofibroblast activation. [ABSTRACT FROM AUTHOR]

Interleukin-11 (IL-11) has recently been identified as a critical profibrotic cytokine, and IL-11 signaling pathway via IL-11Rα and GP130 receptors has been shown to be a promising therapeutic target for the treatment of fibrotic diseases. Herein, we devised two kinds of IL-11 dimer with receptor-biased binding ability through site-specific crosslinking at the interface involving GP130 binding and signaling, aiming to explore their therapeutic potentials for bleomycin-induced pulmonary fibrosis in mice. A single cysteine mutation at site W147 of human IL-11 (IL-11 W147C) was conducted for site-specific crosslinking. The ability of GP130 to bind to IL-11 W147C dimer was substantially weakened by cysteine-based dimerization, while the ability of IL-11 W147C dimer to bind to IL-11Rα was almost entirely preserved or even enhanced. The IL-11 W147C dimer potently inhibited TF-1 cell proliferation and TGF-β1-induced human lung fibroblast differentiation into myofibroblasts. We also showed that dimerization substantially extended the circulation time of IL-11 W147C dimer in healthy rats. Subcutaneous administration of IL-11 W147C dimer significantly reduced extracellular matrix deposition, preserved alveolar architecture and alleviated pulmonary fibrosis development in mice. The findings of this study may provide a general strategy for the design of cytokine-based receptor-biased antagonists and agonists targeting these multifaceted signaling pathways., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Idiopathic pulmonary fibrosis (IPF) is an irreversible and life-threatening lung disease of unknown etiology presenting only a few treatment options. TGF-β signaling orchestrates a cascade of events driving pulmonary fibrosis (PF). Notably, recent research has affirmed the augmentation of TGF-β receptor (TβR) signaling via HSP90 activation. HSP90, a molecular chaperone, adeptly stabilizes and folds TβRs, thus intricately regulating TGF-β1 signaling. Our investigation illuminated the impact of alvespimycin, an HSP90 inhibitor, on TGF-β-mediated transcriptional responses by inducing destabilization of TβRs. This outcome stems from the explicit interaction of TβR subtypes I and II with HSP90, where they are clients of this cellular chaperone. It is worth noting that regulation of proteasome-dependent degradation of TβRs is a critical standpoint in the termination of TGF-β signal transduction. Oleuropein, the principal bioactive compound found in Olea europaea, is acknowledged for its role as a proteasome activator. In this study, our aim was to explore the efficacy of a combined therapy involving oleuropein and alvespimycin for the treatment of PF. We employed a PF rat model that was induced by intratracheal bleomycin infusion. The application of this dual therapy yielded a noteworthy impediment to the undesired activation of TGF-β/mothers against decapentaplegic homologs 2 and 3 (SMAD2/3) signaling. Consequently, this novel combination showcased improvements in both lung tissue structure and function while also effectively restraining key fibrosis markers such as PDGF-BB, TIMP-1, ACTA2, col1a1, and hydroxyproline. On a mechanistic level, our findings unveiled that the antifibrotic impact of this combination therapy likely stemmed from the enhanced degradation of both TβRI and TβRII. In conclusion, the utilization of proteasomal activators in conjunction with HSP90 inhibitors ushers in a promising frontier for the management of PF.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease (ILD). Pulmonary fibroblasts play an important role in the development of IPF. Emerging evidence indicates that pulmonary endothelial cells could be the source of pulmonary fibroblasts through endothelial mesenchymal transition (EndoMT), which contributes to pulmonary fibrosis. EndoMT is a complex process in which endothelial cells lose their expression of endothelial markers and give rise to the characteristics of mesenchymal cells, including morphological fibroblast-like change and the expression of mesenchymal markers, which result in cardiac, renal, and dermal fibroses. Furthermore, EndoMT inhibition attenuates pulmonary fibrosis. Herein, we demonstrate that nintedanib, a tyrosine kinase receptor inhibitor, ameliorated murine bleomycin (BLM)-induced pulmonary fibrosis and suppressed the in vivo and in vitro models of EndoMT. We demonstrated that the activity of focal adhesion kinase (FAK), a key EndoMT regulator, increased in murine lung tissues and human pulmonary microvascular endothelial cells after BLM stimulation. Nintedanib treatment inhibited BLM-induced FAK activation and thus suppressed both in vivo and in vitro BLM-induced EndoMT. Importantly, we found that the VEGF/FAK signaling pathway was involved in nintedanib regulating EndoMT. These novel findings help us understand the mechanism and signaling pathway of EndoMT to further develop more efficacious drugs for IPF treatment. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing interstitial pneumonia of unknown cause. No therapeutic modalities can reverse or stop its ever-deteriorating course. The stimulation of lung innate immunity using bacterial lysates was found to protect against lethal pulmonary infection. Hence, this study aimed to explore whether the immune-stimulating enhancement by pretreatment with bacterial lysates led to protection against bleomycin-induced pulmonary fibrosis. C57BL/6 mice were randomly divided into 4 groups with 20 mice in each group. The mice were exposed to an aerosolized mixture of polyvalent bacterial lysates (PVBL) or phosphate-buffered saline (PBS) three times on separate days. Twenty-four hours after the last exposure, the lungs were intratracheally infused with bleomycin (BLM) or normal saline (NS). The pulmonary morphology, Ashcroft's scale of pulmonary fibrosis, and levels of pro-inflammatory cytokines such as interferon (IFN)-γ and interleukin (IL)-4 were evaluated 14 days after the intratracheal infusion. The exposure to PVBL did not induce any discernible structural abnormalities in the lungs, while the IFN-γ/IL-4 ratio increased. BLM-induced pulmonary fibrosis was associated with an overwhelming downregulation of IFN-γ and IL-4 expression. Pre-exposure to PVBL protected against BLM-induced pulmonary fibrosis, which was demonstrated by a greater reduction of Ashcroft's fibrotic score and a greater decrease in the hydroxyproline level in the lungs. Although the PVBL pre-exposure did not restore the BLM-induced downregulation of IL-4 and IFN-γ levels, the IFN-γ/IL-4 ratio was still maintained greater than the animals with BLM infusion. BLM-induced murine pulmonary fibrosis is associated with downregulation of IFN-γ and IL-4 levels. Pre-exposure to the aerosolized PVBL protects against BLM-induced pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

Insulin-like growth factor (IGF) signaling controls the development and growth of many organs, including the lung. Loss of function of Igf1 or its receptor Igf1r impairs lung development and leads to neonatal respiratory distress in mice. Although many components of the IGF signaling pathway have shown to be dysregulated in idiopathic pulmonary fibrosis (IPF), the expression pattern of such components in different cellular compartments of the developing and/or fibrotic lung has been elusive. In this study, we provide a comprehensive transcriptional profile for such signaling components during embryonic lung development in mice, bleomycin-induced pulmonary fibrosis in mice and in human IPF lung explants. During late gestation, we found that Igf1 is upregulated in parallel to Igf1r downregulation in the lung mesenchyme. Lung tissues derived from bleomycin-treated mice and explanted IPF lungs revealed upregulation of IGF1 in parallel to downregulation of IGF1R, in addition to upregulation of several IGF binding proteins (IGFBPs) in lung fibrosis. Finally, treatment of IPF lung fibroblasts with recombinant IGF1 led to myogenic differentiation. Our data serve as a resource for the transcriptional profile of IGF signaling components and warrant further research on the involvement of this pathway in both lung development and pulmonary disease. [ABSTRACT FROM AUTHOR]

Insulin-like growth factor (IGF) signaling controls the development and growth of many organs, including the lung. Loss of function of Igf1 or its receptor Igf1r impairs lung development and leads to neonatal respiratory distress in mice. Although many components of the IGF signaling pathway have shown to be dysregulated in idiopathic pulmonary fibrosis (IPF), the expression pattern of such components in different cellular compartments of the developing and/or fibrotic lung has been elusive. In this study, we provide a comprehensive transcriptional profile for such signaling components during embryonic lung development in mice, bleomycin-induced pulmonary fibrosis in mice and in human IPF lung explants. During late gestation, we found that Igf1 is upregulated in parallel to Igf1r downregulation in the lung mesenchyme. Lung tissues derived from bleomycin-treated mice and explanted IPF lungs revealed upregulation of IGF1 in parallel to downregulation of IGF1R, in addition to upregulation of several IGF binding proteins (IGFBPs) in lung fibrosis. Finally, treatment of IPF lung fibroblasts with recombinant IGF1 led to myogenic differentiation. Our data serve as a resource for the transcriptional profile of IGF signaling components and warrant further research on the involvement of this pathway in both lung development and pulmonary disease.

Background: Pulmonary 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., Methods: The 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., Results: The 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., Conclusions: The 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., (© 2024 The Authors. Published by Elsevier B.V. on behalf of Chang Gung University. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

New Findings: •What is the central question of this study?The aim was to explore the effects and underlying mechanisms of H2 on bleomycin‐induced pulmonary fibrosis.•What are the main findings and its importance?Our results indicate that, in bleomycin‐induced pulmonary fibrosis, H2 inhalation attenuated oxidative stress and reversed the pulmonary epithelial‐to‐mesenchymal transition process by reducing reactive oxygen species production and inhibiting the expression of transforming growth factor‐β1, α‐smooth muscle actin and collagen I to improve fibrotic injury and exert anti‐fibrogenic effects. Thus, H2 inhalation has promising therapeutic potential as a useful adjuvant treatment for patients with idiopathic pulmonary fibrosis, which deserves further study and evaluation. Hydrogen (H2) can protect against tissue damage. The effect of H2 inhalation therapy on the pathogenesis of pulmonary fibrosis remains unknown. This study was designed to explore the effects and underlying mechanisms of H2 inhalation on bleomycin (BLM)‐induced pulmonary fibrosis. A rat model of pulmonary fibrosis was established with BLM. Rats were randomly divided into control and H2 inhalation groups. Haematoxylin and Eosin staining and Mason's Trichrome staining were performed to evaluate pulmonary fibrosis injury, inflammatory cell infiltration, structural disorder and collagen deposition. qRT‐PCR and western blot assays were used to determine the expression of TNF‐α, TGF‐β1, α‐SMA, E‐cadherin, N‐cadherin, vimentin, VEGF and collagen type I at both mRNA and protein levels. The contents of reactive oxygen species, TGF‐β1, TNF‐α, malondialdehyde and hydroxyproline were determined with biochemical test kits or ELISA kits. Bleomycin‐stimulated rats exhibited typical symptoms of pulmonary fibrosis, which featured an increase in collagen deposition, alveolitis, fibrosis and parenchymal structural disorder in the lung. However, BLM‐induced oxidative stress was attenuated by H2 inhalation therapy, which reduced the contents of reactive oxygen species, malondialdehyde and hydroxyproline, enhanced the activity of glutathione peroxidase and decreased the expression of TGF‐β1 and TNF‐α. In addition, H2 inhalation also inhibited BLM‐induced epithelial‐to‐mesenchymal transition by inhibiting TGF‐β1, increasing the expression level of the epithelial cell marker E‐cadherin, and decreasing the expression level of the mesenchymal cell marker vimentin in a time‐dependent manner. In addition, H2 inhalation downregulated α‐SMA expression and suppressed collagen I generation, exerting anti‐fibrogenic effects. Hydrogen inhalation therapy attenuates BLM‐induced pulmonary fibrosis by inhibiting TGF‐β1, relevant oxidative stress and epithelial‐to‐mesenchymal transition. [ABSTRACT FROM AUTHOR]

The bleomycin-induced pulmonary fibrosis mouse model is commonly used in idiopathic pulmonary fibrosis research, but its cellular and molecular changes and efficiency as a model at the molecular level are not fully understood. In this study, we used spatial transcriptome technology to investigate the cellular and molecular changes in the lungs of bleomycin-induced pulmonary fibrosis mouse models. Our analyses revealed cell dynamics during fibrosis in epithelial cells, mesenchymal cells, immunocytes, and erythrocytes with their spatial distribution available. We confirmed the differentiation of the alveolar type II (AT2) cell type expressing Krt8 , and we inferred their trajectories from both the AT2 cells and club cells. In addition to the fibrosis process, we also noticed evidence of self-resolving, especially to identify possible self-resolving related genes, including Prkca . Our findings provide insights into the cellular and molecular mechanisms underlying fibrosis resolution and represent the first spatiotemporal transcriptome dataset of the bleomycin-induced fibrosis mouse model., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Context: Myrtle (Myrtus communis L) has been used widely in traditional medicine for different respiratory disorders. Idiopathic pulmonary fibrosis (IPF) is an inflammatory disease characterized by progressive loss of lung function with poor prognosis. The pathogenesis of disease has not been completely elucidated, but probably persistent epithelial damages are involved. Objective: Evaluation of biochemical and histopathological effect of preventive and therapeutic doses of myrtle against bleomycin (BLM)-induced pulmonary fibrosis (PF) in animal model. Materials and methods: Methanolic extract of M. communis was prepared by maceration method. Total flavonoid content was determined and experimentally PF was induced in rat with intratracheal instillation of a single dose of BLM (5 mg/kg) only on day 0. Myrtle antifibrotic effect was evaluated as preventive (50 mg/kg/day, intraperitoneal (i.p.) injection, from day 0-13) and therapeutic agent (50 mg/kg, i.p., from day 14-27) in comparison with methyl prednisolone (M-pred) (4 mg/kg, i.p. for 14 days). Results: Parenchymal inflammation and fibrotic changes significantly were reduced by myrtle and M-pred. Significant decrease in hydroxyproline content and lipid peroxidation were observed in animals receiving myrtle extract while catalase activity was increased by myrtle. Improvement in inflammation and fibrosis was observed in myrtle group especially in the early phase of fibrosis (preventive regime). Discussion and conclusion: Myrtle extract effectively inhibited the inflammation and fibrosis of lung parenchyma in both preventive and therapeutic methods. This effect might be due to the reduction of tissue inflammation and inhibition of oxidative stress. More studies are being carried out to find main mechanisms and separation of active compounds. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is the most widely studied interstitial lung disease. IPF eventually leads to respiratory insufficiency, lung cancer, and death. Carvedilol (CAR) is a third-generation β-adrenergic receptor antagonist with an α1-blocking effect. CAR demonstrates antifibrotic activities in various experimental models of organ fibrosis. Aims: This work is designed to explore the possible alleviating effects of CAR on bleomycin (BLM)-induced lung fibrosis in rats. Main methods: The BLM rat model of lung fibrosis was achieved by intratracheal delivery of a single dose of 5 mg/kg of BLM. Seven days following BLM injection, either prednisolone or CAR was orally administered at doses of 10 mg/kg once daily for 21 days to the rats. The actions of CAR were evaluated by lung oxidant/antioxidant parameters, protein concentration and total leucocyte count (TLC) in bronchoalveolar lavage fluid (BALF), fibrosis regulator-related genes along with the coexistent lung histological changes. Key findings: CAR effectively decreased lung malondialdehyde level, increased superoxide dismutase activity, declined both protein concentration and TLC in BALF, downregulated TGF-β1 / α-SMA/Smad2 /3 and STAT3 gene expressions, and repaired the damaged lung tissues. Significance: CAR conferred therapeutic potential against BLM-induced lung fibrosis in rats, at least in part, to its antioxidant, anti-inflammatory, and antifibrotic activities. CAR could be utilized as a prospective therapeutic option in patients with lung fibrosis in clinical practice. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a deadly, progressive lung disease with very few treatment options till now. Bleomycin-induced pulmonary fibrosis (BIPF) is a commonly used mice model in IPF research. TGF-β1 has been shown to play a key role in pulmonary fibrosis (PF). Dendritic cell (DC) acts as a bridge between innate and adaptive immune systems. The coexistence of chronic inflammation sustained by mature DCs with fibrosis suggests that inflammatory phenomenon has key importance in the pathogenesis of pulmonary fibrosis. Here, we investigated the modulation of DCs phenotypic maturation, accumulation in lung tissue, and expression of other lung DC subsets in respect to TGF-β in PF. First, we established BIPF model in mice and blocked TGF-β expression by the use of inhibitor SB431542. Accumulation of lung CD11c+ DCs is significantly higher in both inflammatory and fibrotic phases of the disease but that percentages got reduced in the absence of TGF-β. TGF-β initiates up-regulation of costimulatory molecules CD86 and CD80 in the inflammatory phases of the disease but not so at fibrotic stage. Expression of lung DC subset CD11c+CD103+ is significantly increased in inflammatory phase and also in fibrotic phase of BIPF. Blocking of TGF-β causes decreased expression of CD11c+CD103+ DCs. Another important lung DC subset CD11c+CD11b+ expression is suppressed by the absence of TGF-β after bleomycin administration. CD11c+CD103+ DCs might have anti-inflammatory as well as anti-fibrotic nature in PF. All these data demonstrate differential modulation of CD11c+ lung DCs by TGF-β in experimental PF. [ABSTRACT FROM AUTHOR]

Background: Myofibroblasts derived from fibroblasts in the pathogenesis of pulmonary fibrosis causes excessive and disordered deposition of matrix proteins, including collagen V, which can cause a Th17-mediated immune response and lead to apoptosis. However, whether the intrinsic ability of lung FBs to produce the matrix depends on their site-specific variations is not known. Aim: To investigate the link between Th17 and collagen V that maintains pulmonary remodeling in the peripheral lung microenvironment during the late stage of experimental pulmonary fibrosis. Methods: Young male mice including wild Balb/c mice (BALB, n = 10), wild C57 Black/6J mice (C57, n = 10) and IL-17 receptor A knockout mice (KO, n = 8), were sacrificed 21 days after treatment with bleomycin. Picrosirius red staining, immunohistochemistry for IL-17-related markers and “in situ” detection of apoptosis, immunofluorescence for collagen types I and V, primary cell cultures from tissue lung explants for RT-PCR and electron microscopy were used. Results: The peripheral deposition of extracellular matrix components by myofibroblasts during the late stage is maintained in C57 mice compared with that in Balb mice and is not changed in the absence of IL-17 receptor A; however, the absence of IL-17 receptor A induces overexpression of type V collagen, amplifies the peripheral expression of IL-17 and IL-17-related cytokines and reduces peripheral lung fibroblast apoptosis. Conclusion: A positive feedback loop between the expression patterns of collagen V and IL-17 may coordinate the maintenance of peripheral collagen I in the absence of IL-17 receptor A in fibrosis-susceptible strains in a site-specific manner. [ABSTRACT FROM AUTHOR]

Secretoglobin (SCGB) 3A2 is a member of the SCGB gene superfamily of small secreted proteins, predominantly expressed in lung airways. We hypothesize that human SCGB3A2 may exhibit anti-inflammatory, growth factor, and antifibrotic activities and be of clinical utility. Recombinant human SCGB3A2 was expressed, purified, and bio-chemically characterized as a first step to its development as a therapeutic agent in clinical settings. Human SCGB3A2, as well as mouse SCGB3A2, readily formed a dimer in solution and exhibited novel phospholipase A2 inhibitory activity. This is the first demonstration of any quantitative biochemical measurement for the evaluation of SCGB3A2 protein. In the mouse as an experimental animal, human SCGB3A2 exhibited growth factor activity by promoting embryonic lung development in both ex vivo and in vivo systems and antifibrotic activity in the bleomycin-induced lung fibrosis model. The results suggested that human SCGB3A2 can function as a growth factor and an antifibrotic agent in humans. When SCGB3A2 was administered to pregnant female mice through the tail vein, the protein was detected in the dam's serum and lung, as well as the placenta, amniotic fluids, and embryonic lungs at 10 min postadministration, suggesting that SCGB3A2 readily crosses the placenta. The results warrant further development of recombinant SCGB3A2 as a therapeutic agent in treating patients suffering from lung diseases or preterm infants with respiratory distress. [ABSTRACT FROM AUTHOR]

Ovarian yolk sac tumor (YST) is a highly aggressive malignancy arising in young women. Chemotherapy has dramatically improved the prognosis, and bleomycin, etoposide, and cisplatin (BEP) combination chemotherapy appears to be the most effective combination regimen. A 23-year-old woman was admitted to our hospital with worsening abdominal distention and a lower abdominal mass. She was diagnosed with a stage IIIc pure YST of the right ovary, and right salpingo-oophorectomy was performed; there were numerous disseminated peritoneal tumors within the abdominal cavity. A few days postoperatively, massive ascites developed, and right hydronephrosis occurred. Chemotherapy with BEP was started, and after 24 h of administration, oliguria and tumor lysis syndrome (TLS) developed. Continuous hemodiafiltration was started, and hemodialysis was initiated following full-dose standard cisplatin and etoposide on days 2-5 of the 1st cycle. After the electrolyte abnormalities and the elevation of creatinine became normal, the patient received an additional three cycles of BEP and achieved complete remission. However, she also suffered from severe non-hematological toxicities, including grade 3 left ventricular dysfunction and grade 4 pulmonary fibrosis. In the case of rapidly progressing and high-volume YST treated with BEP chemotherapy, special attention should be paid to bleomycin-induced pulmonary toxicity following TLS. Further study is required to optimize drug exposure to ensure efficacy and reduce the risk of side effects in this population. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing lung disease that is caused by the dysregulation of alveolar epithelial type II cells (AEC II). The mechanisms involved in the progression of IPF remain incompletely understood, although the immune response accompanied by p38 mitogen-activated protein kinase (MAPK) activation may contribute to some of them. This study aimed to examine the association of p38 activity in the lungs with bleomycin (BLM)-induced pulmonary fibrosis and its transcriptomic profiling. Accordingly, we evaluated BLM-induced pulmonary fibrosis during an active fibrosis phase in three genotypes of mice carrying stepwise variations in intrinsic p38 activity in the AEC II and performed RNA sequencing of their lungs. Stepwise elevation of p38 signaling in the lungs of the three genotypes was correlated with increased severity of BLM-induced pulmonary fibrosis exhibiting reduced static compliance and higher collagen content. Transcriptome analysis of these lung samples also showed that the enhanced p38 signaling in the lungs was associated with increased transcription of the genes driving the p38 MAPK pathway and differentially expressed genes elicited by BLM, including those related to fibrosis as well as the immune system. Our findings underscore the significance of p38 MAPK in the progression of pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

Background: Idiopathic Pulmonary Fibrosis (IPF) is a chronic and progressive respiratory disease characterized by the destruction of the alveolar structure. In pulmonary fibrosis, aerosolized drugs are easily transferred to the systemic circulation via leakage through the injured alveolar epithelium. Therefore, pulmonary drug delivery systems for sustained distribution in fibrotic lungs are needed., Objective: We evaluated the intrapulmonary pharmacokinetics of aerosolized liposomes as pulmonary drug delivery systems in mice with bleomycin-induced pulmonary fibrosis., Methods: The aerosolized liposomal formulations and solutions of model compounds, including indocyanine green and 6-carboxyfluorescein (6-CF), were intrapulmonary administered to mice with bleomycin-induced pulmonary fibrosis. In vivo imaging for indocyanine green and 6-CF measurements in lung tissues and plasma were performed. Additionally, in vitro permeation experiments using NCI-H441 cell monolayers as a model of alveolar epithelial cells were performed., Results: The fluorescence signals of indocyanine green following the administration of liposomal formulations were observed longer in the lungs than those in solution-treated mice. Compared with the solution, the 6-CF concentrations in lung tissues after the administration of liposomal formulations were determined higher, whereas those in the plasma were lower. 6-CF permeability was significantly increased by transforming growth factor-β1 in NCI-H441 cell monolayers treated with the solution but unchanged in the presence of the liposomal formulation., Conclusion: The aerosolized liposomal formulation can prevent enhanced drug transfer from fibrotic lungs into the systemic circulation via the injured alveolar epithelium. This system may be useful for the sustained distribution of anti-fibrotic agents in fibrotic lungs and the optimization of IPF therapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Endogenous glutamate (Glu) release and N-methyl-d-aspartate (NMDA) receptor (NMDAR) activation are associated with lung injury in different animal models. However, the underlying mechanism is unclear. Bone marrow-derived mesenchymal stem cells (BM-MSCs), which show potential use for immunomodulation and tissue protection, play a protective role in pulmonary fibrosis (PF) process. Here, we found the increased Glu release from the BM cells of bleomycin (BLM)-induced PF mice in vivo. BLM stimulation also increased the extracellular Glu in BM-MSCs via the antiporter system x c - in vitro. The gene expression of each subunit of NMDAR was detected in BM-MSCs. NMDAR activation inhibited the proliferation, migration, and paracrine function of BM-MSCs in vitro. BM-MSCs were derived from male C57BL/6 mice, transfected with lentiviral vectors carrying the enhanced green fluorescence protein gene, pretreated with NMDA, and transplanted into the female recipient mice that were intratracheally injected with BLM to induce PF. Transplantation of NMDA-pretreated BM-MSCs significantly aggravated PF as compared with that in the normal BM-MSCs transplantation group. The sex determination gene Y chromosome and green fluorescence protein genes of BM-MSCs were detected to observe BM-MSCs homing in the fibrotic lungs. Moreover, NMDAR activation inhibited BM-MSC migration by downregulating the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 signaling axis. NMDAR activation aggravated the transforming growth factor-β1-induced extracellular matrix production in alveolar epithelial cells and fibroblasts through the paracrine effects of BM-MSCs. In summary, these findings suggested that NMDAR activation-mediated Glu excitotoxicity induced by BLM in BM-MSCs abolished the therapeutic effects of normal BM-MSCs transplantation on BLM-induced PF.

Mesenchymal stem cells (MSCs) are widely considered for treatment of pulmonary fibrosis based on the anti-inflammatory, antifibrotic, antiapoptotic, and regenerative properties of the cells. Recently, elevated levels of oncostatin M (OSM) have been reported in the bronchoalveolar lavage fluid of a pulmonary fibrosis animal model and in patients. In this work, we aimed to prolong engrafted MSC survival and to enhance the effectiveness of pulmonary fibrosis transplantation therapy by using OSM-preconditioned MSCs. OSM-preconditioned MSCs were shown to overexpress type 2 OSM receptor (gp130/OSMRβ) and exhibited high susceptibility to OSM, resulting in upregulation of the paracrine factor, hepatocyte growth factor (HGF). Moreover, OSM-preconditioned MSCs enhanced cell proliferation and migration, attenuated transforming growth factor-β1- or OSM-induced extracellular matrix production in MRC-5 fibroblasts through paracrine effects. In bleomycin-induced lung fibrotic mice, transplantation of OSM-preconditioned MSCs significantly improved pulmonary respiratory functions and downregulated expression of inflammatory factors and fibrotic factors in the lung tissues. Histopathologic examination indicated remarkable amelioration of the lung fibrosis. LacZ-tagged MSCs were detected in the lung tissues of the OSM-preconditioned MSC-treated mice 18 days after post-transplantation. Taken together, our data further demonstrated that HGF upregulation played an important role in mediating the therapeutic effects of transplanted OSM-preconditioned MSCs in alleviating lung fibrosis in the mice. Stem Cells Translational Medicine 2017;6:1006-1017., (© 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Secretoglobin (SCGB) 3A2 is a member of the SCGB gene superfamily of small secreted proteins, predominantly expressed in lung airways. We hypothesize that human SCGB3A2 may exhibit anti-inflammatory, growth factor, and antifibrotic activities and be of clinical utility. Recombinant human SCGB3A2 was expressed, purified, and biochemically characterized as a first step to its development as a therapeutic agent in clinical settings. Human SCGB3A2, as well as mouse SCGB3A2, readily formed a dimer in solution and exhibited novel phospholipase A2 inhibitory activity. This is the first demonstration of any quantitative biochemical measurement for the evaluation of SCGB3A2 protein. In the mouse as an experimental animal, human SCGB3A2 exhibited growth factor activity by promoting embryonic lung development in both ex vivo and in vivo systems and antifibrotic activity in the bleomycin-induced lung fibrosis model. The results suggested that human SCGB3A2 can function as a growth factor and an antifibrotic agent in humans. When SCGB3A2 was administered to pregnant female mice through the tail vein, the protein was detected in the dam's serum and lung, as well as the placenta, amniotic fluids, and embryonic lungs at 10 min postadministration, suggesting that SCGB3A2 readily crosses the placenta. The results warrant further development of recombinant SCGB3A2 as a therapeutic agent in treating patients suffering from lung diseases or preterm infants with respiratory distress.