Intratracheal Bleomycin Research Articles

Bleomycin-Induced Fibrosis and the Effectiveness of Centella Asiatica as a Treatment.

Plant treatment has been used for thousands of years and has been proven to treat acute and chronic diseases. The function of the traditional plant Centella asiatica is as an antimicrobial agent, anticancer, antioxidant, and therapeutic gene in healing wounds and inflammation. Lung fibrosis caused by bleomycin can develop into chronic lung disease, which ends in tissue death if not treated immediately. The purpose of this study is to predict and explain the impact of Centella asiatica extract on model rats exposed to bleomycin in their lungs as a treatment or anti-fibrinolysis. This research is an analytical study with a randomized in-vivo experimental design divided into 3 groups of 5 male Wistar rats aged 10 weeks. Negative control group (K) with intratracheal induction of bleomycin alone. The positive group was given intratracheal bleomycin 4 mg/kg/BB on days 0 and 21 and added Centella asiatica induction at 400 mg (P1) on days 15 to 49. The other positive group was given intratracheal bleomycin 4 mg/kg/BB on days 0 and 21 and added Centella asiatica induction at 800 mg (P2) on days 15 to 49. Data were collected according to findings of lung histology analysis of rat samples. In the interalveolar septum group, there was a difference in Masson's Trichrome staining results in groups K and P1 with p<0.05 (p=0.036). However, there was no difference in histopathological staining results in groups K and P2 (p>0.05). The induction of bleomycin 4 mg/kg/BB was proven to cause fibrosis in the lungs of rats, and Centella asiatica extract was used as a treatment. Therefore, further research regarding antifibrotic drugs is hoped to reduce fibrotic areas significantly.

Investigation of Aberrant Basaloid Cells in a Rat Model of Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease (ILD) whose cause and pathogenesis are not yet well understood. Until now, no animal model of lung fibrosis succeeds in recapitulating all IPF features, thus the use of different rodent models is essential for the evaluation and development of new effective pharmacological treatments. Recently, the alveolar epithelial dysfunction has been emphasized in the etiopathogenesis context of IPF. Remarkably, the role of an aberrant basaloid cell type, primarily found in humans and confirmed in mice, seems to be crucial in the establishment and progression of the disease/model. Our work aimed to characterize for the first time this cell population in a rat model of lung fibrosis induced by a double bleomycin (BLM) administration, demonstrating the translational value of the model and its potential use in the testing of effective new drugs. Rats received an intratracheal BLM administration at day 0 and 4. Animals were sacrificed 21 and 28 days post-BLM. The fibrosis evaluation was carried out through histological (Ashcroft score and automatic image analysis) and immunoenzymatic analysis. Immunofluorescence was used for the characterization of the aberrant basaloid cells markers. Lung histology revealed an increase in severe grades of Ashcroft scores and areas of fibrosis, resulting in a rise of collagen deposition at both the analyzed time-points. Immunofluorescence staining indicated the presence of KRT8+ cells in bronchial epithelial cells from both controls (saline, SAL) and BLM-treated animals. Interesting, KRT8+ cells were found exclusively in the fibrotic parenchyma (confirmed by the alpha-smooth muscle actin (α-SMA) staining for myofibroblasts) of BLM-treated animals. Moreover, KRT8+ cells co-expressed markers as Prosurfactant protein C (Pro-SPC) and Vimentin, suggesting their intermediate state potentially originating from alveolar type II (AT2) cells, and participating to the abnormal epithelial-mesenchymal crosstalk. Previous preclinical studies demonstrated the presence of KRT8+ aberrant basaloid-like cells in murine models of lung fibrosis. This work investigated the same cell population in a different rodent (the rat) model of lung fibrosis triggered by a double administration of BLM. Our results provided a further confirmation that, in rats, the intratracheal administration of BLM induced the appearance of a population of cells compatible with the KRT8+ alveolar differentiation intermediate (ADI) cells, as described previously in the mouse. This piece of work enforces previous evidence and further support the use of a rat model of BLM resembling the alveolar epithelial dysfunction to evaluate new clinical candidates for development in IPF.

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.

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.

Tetrahedral DNA loaded siCCR2 restrains M1 macrophage polarization to ameliorate pulmonary fibrosis in chemoradiation-induced murine model

Idiopathic pulmonary fibrosis (IPF) is a chronic lethal disease in the absence of demonstrated efficacy for preventing progression. Although macrophage-mediated alveolitis is determined to participate in myofibrotic transition during disease development, the paradigm of continuous macrophage polarization is still under-explored due to lack of proper animal models. Here, by integrating 2.5U/kg intratracheal Bleomycin administration and 10Gy thorax irradiation at day 7, we generated a murine model with continuous alveolitis-mediated fibrosis, which mimics most of the clinical features of our involved IPF patients. In combination with data from scRNA-seq of patients and a murine IPF model, a decisive role of CCL2/CCR2 axis in driving M1 macrophage polarization was revealed, and M1 macrophage was further confirmed to boost alveolitis in leading myofibroblast activation. Multiple sticky-end tetrahedral framework nucleic acids conjunct with quadruple ccr2-siRNA (FNA-siCCR2) was synthesized in targeting M1 macrophages. FNA-siCCR2 successfully blocked macrophage accumulation in pulmonary parenchyma of the IPF murine model, thus preventing myofibroblast activation and leading to the disease remitting. Overall, our studies lay the groundwork to develop a novel IPF murine model, reveal M1 macrophages as potential therapeutic targets, and establish new treatment strategy by using FNA-siCCR2, which are highly relevant to clinical scenarios and translational research in the field of IPF.

Adenophorae Radix Reduces Fibrosis in a Bleomycin-Induced Pulmonary Fibrosis Mouse Model

Background: A screening study investigated the effects of lung-moistening herbal medicines on a bleomycin-induced pulmonary fibrosis mouse model and reported that the efficacy of Adenophorae Radix (ADR) was higher than that of other remedies. Objectives: This study aimed to investigate the antifibrotic efficacy and mechanism of action of ADR in a mouse model of bleomycin-induced pulmonary fibrosis. Materials and Methods: ADR water extract was prepared and orally administered at doses of 30, 100, and 300 mg/kg for 10 days after intratracheal bleomycin instillation. Changes in the body weight and lung histology were assessed. In addition, the quantity of inflammatory cells in the bronchoalveolar lavage fluid (BALF) and the level of transforming growth factor-β and α-smooth muscle actin (α-SMA) were measured. Results: ADR treatment decreased the degree of histological fibrosis based on the Ashcroft score and the number of inflammatory cells in BALF. Moreover, α-SMA level in the lung tissue decreased. ADR at 300 mg/kg resulted in the most significant reduction in the measured indicators. Conclusion: The results indicate that the administration of 300 mg/kg ADR attenuates pulmonary fibrosis in an animal model of idiopathic pulmonary fibrosis; thus, ADR can be considered as a candidate treatment for lung fibrosis.

BCL-2 Modulates IRE1α Activation to Attenuate Endoplasmic Reticulum Stress and Pulmonary Fibrosis.

BCL-2 family members are known to be implicated in survival in numerous biological settings. Here, we provide evidence that in injury and repair processes in lungs, BCL-2 mainly acts to attenuate endoplasmic reticulum (ER) stress and limit extracellular matrix accumulation. Days after an intratracheal bleomycin challenge, mice lose a fraction of their alveolar type II epithelium from terminal ER stress driven by activation of the critical ER sensor and stress effector IRE1α. This fraction is dramatically increased by BCL-2 inhibition, because IRE1α activation is dependent on its physical association with the BCL-2-proapoptotic family member BAX, and we found BCL-2 to disrupt this association invitro. In vivo, navitoclax (a BCL-2/BCL-xL inhibitor) given 15-21 days after bleomycin challenge evoked strong activation of IRE-1α in mesenchymal cells and markers of ER stress, but not apoptosis. Remarkably, after BCL-2 inhibition, bleomycin-exposed mice demonstrated persistent collagen accumulation at Day 42, compared with resolution in controls. Enhanced fibrosis proved to be due to the RNAase activity of IRE1α downregulating MRC2 mRNA and protein, a mediator of collagen turnover. The critical role of MRC2 was confirmed in precision-cut lung slice cultures of Day-42 lungs from bleomycin-exposed wild-type and MRC2 null mice. Soluble and tissue collagen accumulated in precision-cut lung slice cultures from navitoclax-treated, bleomycin-challenged mice compared with controls, in a manner nearly identical to that of challenged but untreated MRC2 null mice. Thus, apart from mitochondrial-based antiapoptosis, BCL-2 functions to attenuate ER stress responses, fostering tissue homeostasis and injury repair.

Qualitative Histopathological Analysis of Bleomycin-Induced Lung Injury

Recent evidence has suggested that the accumulation of cholesterol-laden “fatty macrophages” in the lung may play a key role in the presentation of ALI 1, a condition that affects nearly 200,000 people per year in the United States. Studying the pathways of these cells suggests that preventing the formation and build-up of these macrophages could prevent many of the characteristic symptoms of acute lung injury. Specifically, this experiment aims to explore the effect that ACAT1 inhibition by K604 has on fatty macrophage formation and outcomes of ALI. To test this, different groups of mice were administered either PBS (as control), ITB (intratracheal bleomycin), PBS + K604, or ITB + K604. After the mice were sacrificed, the left lung lobes were extracted, inflated, embedded, and sectioned for microscopic scanning and analysis with the ImageJ software. The lung tissue was characterized using 3 unbiased parameters: alveolar wall thickness (µm), cell infiltration (number of nuclei), and percent white space. Simultaneously, a cholesterol assay was performed using cells extracted from mouse bronchoalveolar lavage (BAL). The results show that K604 was successful at preventing ITB-induced increases in cell infiltration, consolidation, and alveolar thickening as well as lipid accumulation in macrophages by limiting the formation of cholesterol esters through ACAT1 inhibition. In this respect, this experiment shows that K604 might be a viable pharmaceutical target in the treatment of ALI.

Direct assessment of nitrative stress in lipid environments: Applications of a designer lipid-based biosensor for peroxynitrite

Lipid membranes and lipid-rich organelles are targets of peroxynitrite (ONOO-), a highly reactive species generated under nitrative stress. We report a membrane-localized phospholipid (DPPC-TC-ONOO-) that allows the detection of ONOO- in diverse lipid environments: biomimetic vesicles, mammalian cell compartments, and within the lung lining. DPPC-TC-ONOO- and POPC self-assemble to membrane vesicles that fluorogenically and selectively respond to ONOO-. DPPC-TC-ONOO-, delivered through lipid nanoparticles, allowed for ONOO- detection in the endoplasmic reticulum upon cytokine-induced nitrative stress in live mammalian cells. It also responded to ONOO- within lung tissue murine models upon acute lung injury. We observed nitrative stress around bronchioles in precision cut lung slices exposed to nitrogen mustard and in pulmonary macrophages following intratracheal bleomycin challenge. Results showed that DPPC-TC-ONOO- functions specifically toward iNOS, a key enzyme modulating nitrative stress, and offers significant advantages over its hydrophilic analog in terms of localization and signal generation.

Longitudinal x-ray based lung function measurement for monitoring Nintedanib treatment response in a mouse model of lung fibrosis

Lung fibrosis (LF) is a chronic progressive, incurable, and debilitating condition of the lung, which is associated with different lung disease. Treatment options are still sparse. Nintedanib, an oral tyrosine kinase inhibitor, significantly slows the LF progression. However, there is a strong need of further research and the development of novel therapies. In this study, we used a correlative set-up that combines X-ray based lung function (XLF) with microCT and whole body plethysmography (WBP) for a comprehensive functional and structural evaluation of lung fibrosis (LF) as well as for monitoring response to orally administered Nintedanib in the mouse model of bleomycin induced LF. The decline in lung function as early as one week after intratracheal bleomycin instillation was reliably detected by XLF, revealing the lowest decay rate in the LF mice compared to healthy ones. Simultaneously performed microCT and WBP measurements corroborated XLF findings by exhibiting reduced lung volume V^{insp}_{mu CT} and tidal volume TV_{WBP}. In LF mice XLF also revealed profound improvement in lung function one week after Nintedanib treatment. This positive response to Nintedanib therapy was further substantiated by microCT and WBP measurements which also showed significantly improved V^{insp}_{mu CT} and TV_{WBP} in the Nintedanib treated mice. By comparing the XLF data to structural features assessing the extent of fibrosis obtained by ex-vivo high-resolution synchrotron radiation-based imaging and classical histology we demonstrate that: (1) a simple low dose x-ray measurement like XLF is sensitive enough to pick up treatment response, (2) Nintedanib treatment successfully improved lung function in a bleomycin induced LF mouse model and (3) differences between the fully restored lung function and the partially reduced fibrotic burden compared to healthy and untreated mice. The presented analysis pipeline underlines the importance of a combined functional and anatomical readout to reliably measure treatment response and could easily be adapted to other preclinical lung disease models.

MicroRNA-124-3p alleviates repetitive bleomycin induced idiopathic pulmonary fibrosis in mouse by repressing Wnt/β-catenin signaling component AXIN1

Background&#x0D; MicroRNA (miR)-124-3p is a crucial player in the transforming growth factor β1-induced in vitro fibrogenic differentiation of mesenchymal stem cells. In the current study we aimed to further verify the in vivo role of miR-124-3p in a mouse model of idiopathic pulmonary fibrosis (IPF).&#x0D; Methods&#x0D; Mouse IPF model was established using repetitive intratracheal bleomycin (BLM) dosing, followed by in vivo delivery of miR-124-3p. Masson’s trichrome staining, Hematoxyline-eosin (H&amp;E), as well as modified Ashcroft score, were performed on lung tissues to assess extent of pulmonary fibrosis. Collagen deposition was examined using hydroxyproline assay. Inflammatory cell counts were evaluated in the bronchoalveolar Lavage (BAL) fluid. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to assess apoptosis in lung tissues.&#x0D; Results&#x0D; Repetitive BLM injection induced elevated Fibrosis score and collagen deposition, elevated numbers of inflammatory cells in the BAL fluid, and promoted in lung tissues of mice. MiR-124-3p, to a considerable extent, reversed the BLM-induced IPF symptoms in terms of fibrosis score and collagen deposition in the lung tissues, reduced the BLM-elevated inflammatory cells in the BAL fluid and the percentage of BLM-induced apoptotic cells in lung tissues. AXIN1, a pivotal component in Wnt signaling, was also significantly inhibited by miR-124-3p in the experimental mice.&#x0D; Conclusion&#x0D; MiR-124-3p serves as a therapeutic target in the mouse model of IPF by repressing Wnt/β-catenin signaling component AXIN1, and holds great clinical potential in molecular therapies to treat human IPF patients.

Optimization of an Allysine-Targeted PET Probe for Quantifying Fibrogenesis in a Mouse Model of Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a destructive lung disease with a poor prognosis, an unpredictable clinical course, and inadequate therapies. There are currently no measures of disease activity to guide clinicians making treatment decisions. The aim of this study was to develop a PET probe to identify lung fibrogenesis using a pre-clinical model of pulmonary fibrosis, with potential for translation into clinical use to predict disease progression and inform treatment decisions. Eight novel allysine-targeting chelators, PIF-1, PIF-2, …, PIF-8, with different aldehyde-reactive moieties were designed, synthesized, and radiolabeled with gallium-68 or copper-64. PET probe performance was assessed in C57BL/6J male mice 2 weeks after intratracheal bleomycin challenge and in naïve mice by dynamic PET/MR imaging and with biodistribution at 90 min post injection. Lung hydroxyproline and allysine were quantified ex vivo and histological staining for fibrosis and aldehyde was performed. In vivo screening of probes identified 68GaPIF-3 and 68GaPIF-7 as probes with high uptake in injured lung, high uptake in injured lung versus normal lung, and high uptake in injured lung versus adjacent liver and heart tissue. A crossover, intra-animal PET/MR imaging study of 68GaPIF-3 and 68GaPIF-7 confirmed 68GaPIF-7 as the superior probe. Specificity for fibrogenesis was confirmed in a crossover, intra-animal PET/MR imaging study with 68GaPIF-7 and a non-binding control compound, 68GaPIF-Ctrl. Substituting copper-64 for gallium-68 did not affect lung uptake or specificity indicating that either isotope could be used. A series of allysine-reactive PET probes with variations in the aldehyde-reactive moiety were evaluated in a pre-clinical model of lung fibrosis. The hydrazine-bearing probe, 68GaPIF-7, exhibited the highest uptake in fibrogenic lung, low uptake in surrounding liver or heart tissue, and low lung uptake in healthy mice and should be considered for further clinical translation.

Alvespimycin Exhibits Potential Anti-TGF-β Signaling in the Setting of a Proteasome Activator in Rats with Bleomycin-Induced Pulmonary Fibrosis: A Promising Novel Approach.

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.

Betulin-rich hydroalcoholic extract of Daphne oleoides attenuates bleomycin-induced pulmonary fibrosis in rat

Background and objectivePulmonary fibrosis (PF) is a chronic and progressive respiratory disease representing the final stage of lung inflammatory disorders. Reactive oxygen species (ROS), an essential factor in the formation and progression of pulmonary fibrosis, are a significant adverse effect of Bleomycin (BLM). Antioxidant activities have been found in Daphne oleoides. In this study, we attempted to explore the function of hydroalcoholic extract of Daphne oleoides (D. oleoides) and Betulin in inhibiting bleomycin (BLM)-induced pulmonary fibrosis in rat". Materials and methodsThe current experimental study used 36 male Wistar rats (180–220). Following a random process, the animals were divided into six groups six (n = 6). Group, I (the control group) received normal saline, while Group II (the hazardous group) received intratracheal BLM (7.5 units per kg). Following the administration of BLM, Groups V and VI received daily doses of vitamin E (500 mg/kg/d, p.o.) and Betulin (10 mg kg/d, p.o.), whereas Groups III and IV received daily doses of Daphne oleoides extract (300 and 600 mg/kg/d, p.o.). Then, blood samples from the hearts of the animals were taken to assess the plasma concentrations of nitric oxide (NO) and malondialdehyde (MDA). Finally, the rats were euthanized, and the lung tissues were taken out for histological analysis and assessments of the levels of lung hydroxyproline (HP), ferric-reducing ability (FRAP), NO, Glutathione Concentration (GSH), thiol content (tSH) and MDA. FindingsElevated lung index, lung hydroxyproline, NO, and MDA plasma levels, and a reduction in total body thiol content (tSH) in the group receiving BLM were evidence of pulmonary toxicity. Treatment with D. oleoides extracts, Betulin, and Vit E, especially at 600 mg/kg, led to a marked reduction in the above parameters compared with the BLM-received group (p < 0.01). Histological Analysis of the BLM-treated group showed a considerable Lung injury with interstitial infiltration, collapsed alveolar spaces, and alveolar septal thickening. These changes were mitigated with D. oleoides 600, Betulin-, and vitamin E. These changes were mitigated with D. oleoides 600, Betulin-, and vitamin E. ConclusionThese findings suggest that D. oleoides and Betulin prevent bleomycin-induced lung fibrosis in rats by decreasing inflammatory and antioxidant markers. Daphne oleoides, therefore, have the potential to be used therapeutically to treat pulmonary fibrosis.

Inhibition of Fatty Acid Amide Hydrolase (FAAH) Regulates NF-kb Pathways Reducing Bleomycin-Induced Chronic Lung Inflammation and Pulmonary Fibrosis.

The deadly interstitial lung condition known as idiopathic pulmonary fibrosis (IPF) worsens over time and for no apparent reason. The traditional therapy approaches for IPF, which include corticosteroids and immunomodulatory drugs, are often ineffective and can have noticeable side effects. The endocannabinoids are hydrolyzed by a membrane protein called fatty acid amide hydrolase (FAAH). Increasing endogenous levels of endocannabinoid by pharmacologically inhibiting FAAH results in numerous analgesic advantages in a variety of experimental models for pre-clinical pain and inflammation. In our study, we mimicked IPF by administering intratracheal bleomycin, and we administered oral URB878 at a dose of 5 mg/kg. The histological changes, cell infiltration, pro-inflammatory cytokine production, inflammation, and nitrosative stress caused by bleomycin were all reduced by URB878. Our data clearly demonstrate for the first time that the inhibition of FAAH activity was able to counteract not only the histological alteration bleomycin-induced but also the cascade of related inflammatory events.

Combination of Niclosamide and Pirfenidone Alleviates Pulmonary Fibrosis by Inhibiting Oxidative Stress and MAPK/Nf-κB and STATs Regulated Genes.

The pathogenesis of pulmonary fibrosis (PF) is extremely complex and involves numerous intersecting pathways. The successful management of PF may require combining multiple agents. There is a growing body of evidence that suggests the potential benefits of niclosamide (NCL), an FDA-approved anthelminthic drug, in targeting different fibrogenesis molecules. This study aimed at investigating the anti-fibrotic potential of NCL alone and in combination with pirfenidone (PRF), an approved drug for PF, in a bleomycin (BLM) induced PF experimental model. PF was induced in rats by intratracheal BLM administration. The effect of NCL and PRF individually and in combination on different histological and biochemical parameters of fibrosis was investigated. Results revealed that NCL and PRF individually and in combination alleviated the histopathological changes, extracellular matrix deposition and myofibroblastic activation induced by BLM. NCL and PRF either individually or in combination inhibited the oxidative stress and subsequent pathways. They modulated the process of fibrogenesis by inhibiting MAPK/NF-κB and downstream cytokines. They inhibited STATs and downstream survival-related genes including BCL-2, VEGF, HIF-α and IL-6. Combining both drugs showed significant improvement in the tested markers in comparison to the monotherapy. NCL, therefore, has a potential synergistic effect with PRF in reducing the severity of PF.

Modulatory effect of D-pinitol on bleomycin-induced pulmonary fibrosis in rats

Objective: To assess the effect of D-pinitol on pulmonary fibrosis induced by bleomycin. Methods: Sprague-Dawley rats received intratracheal bleomycin (6 IU/kg) to induce pulmonary fibrosis, followed by administration of either D-pinitol (5, 10, or 20 mg/kg) or vehicle or methylprednisolone (10 mg/kg) over 28 days after bleomycin administration. Lung function, biochemical parameters, serum biochemistry, mRNA expressions, and histological features were observed. Results: D-pinitol at 10 and 20 mg/kg significantly (P&lt;0.05) attenuated bleomycin-induced bronchoalveolar lavage fluid, decreased myeloperoxidase, nitric oxide, malondialdehyde levels, and increased glutathione and superoxide dismutase level. D-pinitol also improved lung function (enhanced pause, frequency of breathing, expired volume, and tidal volume). Besides, D-pinitol significantly (P&lt;0.05) upregulated Nrf2 and downregulated mRNA expressions of TGF-β, collagen-1, and Smad-3. Furthermore, considerably less inflammation (peribronchial, perivascular, and total), Ashcroft, and interstitial fibrosis scores were observed in the D-pinitol group. Conclusions: D-pinitol exerts its effect against bleomycin-induced pulmonary fibrosis via antioxidative and anti-fibrotic pathways.

A novel quantification method of lung fibrosis based on Micro-CT images developed with the optimized pulmonary fibrosis mice model induced by bleomycin

Background and aimsIdiopathic pulmonary fibrosis (IPF) is a fibrosing lung disease with unknown etiology, leading to cough and dyspnoea, which is also one of the most common sequelae affecting the quality of life of COVID-19 survivors. There is no cure for IPF patients. We aim to develop a reliable IPF animal model with quantification of fibrosis based on Micro-Computer Tomography (micro-CT) images for the new drug discovery, because different bleomycin administration routes, doses, and intervals are reported in the literature, and there is no quantitative assessment of pulmonary fibrosis based on micro-CT images in animal studies. MethodsWe compared three dosages (1.25 mg/kg, 2.5 mg/kg, and 5 mg/kg) of intratracheal bleomycin administration and experiment intervals (14 and 21 days) in C57BL/6 mice by investigating survival rates, pulmonary histopathology, micro-CT, peripheral CD4+ & CD8+ cells, and cytokines. Moreover, a simple and reliable new method was developed for scoring fibrosis in live mice based on Micro-CT images by using Image J software, which transfers the dark sections in pulmonary Micro-CT images to light colors on a black background. ResultsThe levels of hydroxyproline, inflammation cytokine, fibrotic pathological changes, and collagen deposition in the lungs of mice were bleomycin dose-dependent and time-dependent as well as the body weight loss. Based on the above results, the mice model at 21 days after being given bleomycin at 1.25 mg/kg has optimal pulmonary fibrosis with a high survival rate and low toxicity. There is a significant decrease in the light area (gray value at 9.86 ± 0.72) in the BLM mice, indicating that a significant decrease in the alveolar air area was observed in BLM injured mice compared to normal groups (###p < 0.001), while the Pirfenidone administration increased the light area (gray value) to 21.71 ± 2.95 which is close to the value observed in the normal mice (gray value at 23.23 ± 1.66), which is consistent with the protein levels of Col1A1, and α-SMA. Notably, the standard deviations for the consecutive six images of each group indicate the precision of this developed quantitation method for the micro-CT image taken at the fifth rib of each mouse. ConclusionProvided a quantifying method for Micro-CT images in an optimal and repeatable pulmonary fibrosis mice model for exploring novel therapeutic interventions.

Transcriptomic and proteomic profiling of young and old mice in the bleomycin model reveals high similarity.

The most common preclinical, in vivo model to study lung fibrosis is the bleomycin-induced lung fibrosis model in 2- to 3-mo-old mice. Although this model resembles key aspects of idiopathic pulmonary fibrosis (IPF), there are limitations in its predictability for the human disease. One of the main differences is the juvenile age of animals that are commonly used in experiments, resembling humans of around 20 yr. Because IPF patients are usually older than 60 yr, aging appears to play an important role in the pathogenesis of lung fibrosis. Therefore, we compared young (3 months) and old mice (21 months) 21 days after intratracheal bleomycin instillation. Analyzing lung transcriptomics (mRNAs and miRNAs) and proteomics, we found most pathways to be similarly regulated in young and old mice. However, old mice show imbalanced protein homeostasis as well as an increased inflammatory state in the fibrotic phase compared to young mice. Comparisons with published human transcriptomic data sets (GSE47460, GSE32537, and GSE24206) revealed that the gene signature of old animals correlates significantly better with IPF patients, and it also turned human healthy individuals better into "IPF patients" using an approach based on predictive disease modeling. Both young and old animals show similar molecular hallmarks of IPF in the bleomycin-induced lung fibrosis model, although old mice more closely resemble several features associated with IPF in comparison to young animals.

Vinpocetine's immunomodulating, anti-oxidant, anti-inflammatory, ant-ifibrotic, and PDE inhibiting potencies ameliorate bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis (PF) is a global health problem with a high economic burden. Intratracheal administration of bleomycin is the best model that resembles the pathogenesis of PF in humans. Recently, vinpocetine proved to have neuroprotective, cardioprotective, hepatoprotective, anti-aging, and antifibrotic effects through its anti-oxidant, immunomodulating, and anti-inflammatory activities. The present study investigated the antifibrotic potentiality of vinpocetine in a rat model of PF induced by intratracheal bleomycin administration. PF induced by a single intratracheal instillation of 5 mg/kg bleomycin in nine-week-old Wister rats. Oral vinpocetine was used at doses of 5, 10, or 20 mg/kg to treat PF for 21 days immediately after the bleomycin instillation. Vinpocetine dose-dependently ameliorates PF induced by bleomycin administration since vinpocetine effectively restored the normal body weight gain rates, pulmonary architecture, and collagen fiber distribution and suppressed the elevated BALF cell count, lymphocytes and neutrophils percentage, BALF, IL-6, TNF-α, and TGF-β1 levels and LDH activity, lung tissue MDA level, PDE activity, hydroxyproline content, immunohistochemical expression of α-SMA and CD68 positive macrophage, and fibrosis score. Meanwhile, it efficiently augmented the reduced BALF macrophage percentage, IL-10 level, lung tissue GSH level, CAT, and SOD activities. Vinpocetine may propose a new promising agent to manage PF.