Model Of Idiopathic Pulmonary Fibrosis Research Articles

Shenqi Chongcao Formula ameliorates inflammatory response in rats with pulmonary fibrosis by activating the ASS1/src/STAT3 signaling pathway

To observe the effect of Shenqi Chongcao (SQCC) Formula on the ASS1/src/STAT3 signaling pathway in a rat model of lung fibrosis and explore its therapeutic mechanism. A total of 120 male SD rats were divided equally into 5 groups, including a blank control group with saline treatment and 4 groups of rat models of idiopathic pulmonary fibrosis induced by intratracheal instillation of bleomycin. One day after modeling, the rat models were treated with daily gavage of 10 mL/kg saline, SQCC decoction (0.423 g/kg), pirfenidone (10 mL/kg), or intraperitoneal injection of arginine deiminase (ADI; 2.25 mg/kg, every 3 days) for 28 days. After the treatments, the lung tissues of the rats were collected for calculating the lung/body weight ratio, observing histopathology using HE and Masson staining, and analyzing the inflammatory cells in BALF using Giemsa staining. Serum chemokine ligand 2 (CCL2) and transforming growth factor-β1 (TGF-β1) levels were measured with ELISA. The protein expressions of src, p-srcTry529, STAT3, and p-STAT3Try705 and the mRNA expressions of ASS1, src and STAT3 in the lung tissues were detected using Western blotting and RT-qPCR. The neutrophil, macrophage and lymphocyte counts and serum levels of CCL2 and TGF-β1 were significantly lower in SQCC, pirfenidone and ADI treatment groups than in the model group at each time point of measurement (P < 0.05). P-srcTry529 and p-STAT3Try705 protein expression levels and ASS1, src, and STAT3 mRNA in the lung tissues were also significantly lower in the 3 treatment groups than in the model group (P < 0.05). SQCC Formula can alleviate lung fibrosis in rats possibly by activating the ASS1/src/STAT3 signaling pathway in the lung tissues.

A prognostic model of idiopathic pulmonary fibrosis constructed based on macrophage and mitochondria-related genes

BackgroundStudies have shown that mitochondrial function and macrophages may play a role in the development of idiopathic pulmonary fibrosis (IPF). However, the understanding of the interactions and specific mechanisms between mitochondrial function and macrophages in pulmonary fibrosis is still very limited.MethodsTo construct a prognostic model for IPF based on Macrophage- related genes (MaRGs) and Mitochondria-related genes (MitoRGs), differential analysis was performed to achieve differentially expressed genes (DEGs) between IPF and Control groups in the GSE28042 dataset. Then, MitoRGs, MaRGs and DEGs were overlapped to screen out the signature genes. The univariate Cox analysis and the least absolute shrinkage and selection operator (LASSO) algorithm were implemented to achieve key genes. Furthermore, the independent prognostic analysis was employed. The ingenuity pathway analysis (IPA) was employed to further understand the molecular mechanisms of key genes.Next, the immune infiltration analysis was implemented to identify differential immune cells between two risk subgroups.ResultsThere were 4791 DEGs between IPF and Control groups. Furthermore, 26 signature genes were achieved by the intersection processing. Three key genes including ALDH2, MCL1, and BCL2A1 were achieved, and the risk model based on the key genes was created. In addition, a nomogram for survival forecasting of IPF patients was created based on riskScore, Age, and Gender, and we found that key genes were associated with classical pathways including ‘Apoptosis Signaling’, ‘PI3K/AKT Signaling’, and so on. Next, two differential immune cells including Monocytes and CD8 T cells were identified between two risk subgroups. Moreover, we found that MIR29B2CHG and hsa-mir-1-3p could regulate the expression of ALDH2.ConclusionWe achieved 3 key genes including ALDH2, MCL1,, and BCL2A1 associated with IPF, providing a new theoretical basis for clinical treatment 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.

Induced pluripotent stem cells modulate the Wnt pathway in the bleomycin-induced model of idiopathic pulmonary fibrosis

BackgroundThe Wnt signaling pathway has been implicated in the pathogenesis of fibrotic disorders and malignancies. Hence, we aimed to assess the potential of the induced pluripotent stem cells (IPS) in modulating the expression of the cardinal genes of the Wnt pathway in a mouse model of idiopathic pulmonary fibrosis (IPF).MethodsC57Bl/6 mice were randomly divided into three groups of Control, Bleomycin (BLM), and BLM + IPS; the BLM mice received intratracheal instillation of bleomycin, BLM + IPS mice received tail vein injection of IPS cells 48 h post instillation of the BLM; The Control group received Phosphate-buffered saline instead. After 3 weeks, the mice were sacrificed and Histologic assessments including hydroxy proline assay, Hematoxylin and Eosin, and Masson-trichrome staining were performed. The expression of the genes for Wnt, β-Catenin, Lef, Dkk1, and Bmp4 was assessed utilizing specific primers and SYBR green master mix.ResultsHistologic assessments revealed that the fibrotic lesions and inflammation were significantly alleviated in the BLM + IPS group. Besides, the gene expression analyses demonstrated the upregulation of Wnt, β-Catenin, and LEF along with the significant downregulation of the Bmp4 and DKK1 in response to bleomycin treatment; subsequently, it was found that the treatment of the IPF mice with IPS cells results in the downregulation of the Wnt, β-Catenin, and Lef, as well as upregulation of the Dkk1, but not the Bmp4 gene (P values < 0.05).ConclusionThe current study highlights the therapeutic potential of the IPS cells on the IPF mouse model in terms of regulating the aberrant expression of the factors contributing to the Wnt signaling pathway.

The molecular mechanisms of extracellular matrix-derived hydrogel therapy in idiopathic pulmonary fibrosis models

Idiopathic Pulmonary Fibrosis (IPF) is a progressively debilitating lung condition characterized by oxidative stress, cell phenotype shifts, and excessive extracellular matrix (ECM) deposition. Recent studies have shown promising results using decellularized ECM-derived hydrogels produced through pepsin digestion in various lung injury models and even a human clinical trial for myocardial infarction. This study aimed to characterize the composition of ECM-derived hydrogels, assess their potential to prevent fibrosis in bleomycin-induced IPF models, and unravel their underlying molecular mechanisms of action. Porcine lungs were decellularized and pepsin-digested for 48 h. The hydrogel production process, including visualization of protein molecular weight distribution and hydrogel gelation, was characterized. Peptidomics analysis of ECM-derived hydrogel contained peptides from 224 proteins. Probable bioactive and cell-penetrating peptides, including collagen IV, laminin beta 2, and actin alpha 1, were identified. ECM-derived hydrogel treatment was administered as an early intervention to prevent fibrosis advancement in rat models of bleomycin-induced pulmonary fibrosis. ECM-derived hydrogel concentrations of 1 mg/mL and 2 mg/mL showed subtle but noticeable effects on reducing lung inflammation, oxidative damage, and protein markers related to fibrosis (e.g., alpha-smooth muscle actin, collagen I). Moreover, distinct changes were observed in macroscopic appearance, alveolar structure, collagen deposition, and protein expression between lungs that received ECM-derived hydrogel and control fibrotic lungs. Proteomic analyses revealed significant protein and gene expression changes related to cellular processes, pathways, and components involved in tissue remodeling, inflammation, and cytoskeleton regulation. RNA sequencing highlighted differentially expressed genes associated with various cellular processes, such as tissue remodeling, hormone secretion, cell chemotaxis, and cytoskeleton engagement. This study suggests that ECM-derived hydrogel treatment influence pathways associated with tissue repair, inflammation regulation, cytoskeleton reorganization, and cellular response to injury, potentially offering therapeutic benefits in preventing or mitigating lung fibrosis.

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.

SPECT Imaging of Lysyl Oxidase-like 2 in a Model of Idiopathic Pulmonary Fibrosis.

Noninvasive imaging of idiopathic pulmonary fibrosis (IPF) remains a challenge. The aim of this study was to develop an antibody-based radiotracer targeting Lysyl Oxidase-like 2 (LOXL2), an enzyme involved in the fibrogenesis process, for SPECT/CT imaging of pulmonary fibrosis. The bifunctional chelator DOTAGA-PEG4-NH2 was chemoenzymatically conjugated to the murine antibody AB0023 using microbial transglutaminase, resulting in a degree of labeling (number of chelators per antibody) of 2.3. Biolayer interferometry confirmed that the binding affinity of DOTAGA-AB0023 to LOXL2 was preserved with a dissociation constant of 2.45 ± 0.04 nM. DOTAGA-AB0023 was then labeled with 111In and in vivo experiments were carried out in a mice model of progressive pulmonary fibrosis induced by intratracheal administration of bleomycin. [111In]In-DOTAGA-AB0023 was injected in three groups of mice (control, fibrotic, and treated with nintedanib). SPECT/CT images were recorded over 4 days p.i. and an ex vivo biodistribution study was performed by gamma counting. A significant accumulation of the tracer in the lungs of the fibrotic mice was observed at D18 post-bleomycin. Interestingly, the tracer uptake was found selectively upregulated in fibrotic lesions observed on CT scans. Images of mice that received the antifibrotic drug nintedanib from D8 up to D18 showed a decrease in [111In]In-DOTAGA-AB0023 lung uptake associated with a decrease in pulmonary fibrosis measured by CT scan. In conclusion, we report the first radioimmunotracer targeting the protein LOXL2 for nuclear imaging of IPF. The tracer showed promising results in a preclinical model of bleomycin-induced pulmonary fibrosis, with high lung uptake in fibrotic areas, and accounted for the antifibrotic activity of nintedanib.

Exploratory phase 2 study of the novel oral multi-kinase inhibitor TAS-115 in patients with idiopathic pulmonary fibrosis

BackgroundTAS-115, a novel oral multi-kinase inhibitor, showed antifibrotic effects in in vitro and in vivo animal models of idiopathic pulmonary fibrosis (IPF). MethodsIn this exploratory phase 2 study, IPF patients with a percent predicted forced vital capacity (%FVC) decline ≥5% acquired within the previous 6 months were enrolled. Patients were divided into three pre-treatment cohorts, namely, treatment-naïve, pirfenidone, or nintedanib. TAS-115 was administered orally at 200 mg/day with a 5-day on and 2-day off regimen. After 13 weeks of treatment, patients entered a 13-week extension treatment period where the efficacy was evaluated. The primary endpoint was the difference in slope of %FVC decline at Week 13 from baseline. Safety was also evaluated. ResultsBetween June 2018 and July 2019, 46 patients were enrolled, and 30 (65.2%) patients completed the 13-week treatment. Of these, 22 (47.8%) proceeded to extension treatment. For the primary endpoint, TAS-115 treatment lowered the slope of the %FVC decline of 0.0750%/day (95% confidence interval: 0.0341–0.1158%/day) at Week 13. Efficacy was also demonstrated at Week 26. Treatment-related adverse events were reported in 40 (88.9%) patients, but most were manageable by dose reduction, dose interruption, or symptomatic treatment. ConclusionsTAS-115 treatment was effective, assessed using intra-patient change in slope of %FVC decline as a surrogate endpoint in patients with IPF pre-treated with pirfenidone or nintedanib and treatment-naïve patients. TAS-115 showed acceptable tolerability and a manageable safety profile. Trial registrationJapic-Clinical Trials Information, JapicCTI-183898 (first registered: March 15, 2018).

Variations in mechanical stiffness alter microvascular sprouting and stability in a PEG hydrogel model of idiopathic pulmonary fibrosis.

Microvascular remodeling is governed by biomechanical and biochemical cues which are dysregulated in idiopathic pulmonary fibrosis. Understanding how these cues impact endothelial cell-pericyte interactions necessitates a model system in which both variables can be independently and reproducibly modulated. In this study we develop a tunable hydrogel-based angiogenesis assay to study how varying angiogenic growth factors and environmental stiffness affect sprouting and vessel organization. Lungs harvested from mice were cut into 1 mm long segments then cultured on hydrogels having one of seven possible stiffness and growth factor combinations. Time course, brightfield, and immunofluorescence imaging were used to observe and quantify sprout formation. Our assay was able to support angiogenesis in a comparable manner to Matrigel in soft 2 kPa gels while enabling tunability to study the effects of stiffness on sprout formation. Matrigel and 2 kPa groups contained significantly more samples with sprouts when compared to the stiffer 10 and 20 kPa gels. Growth factor treatment did not have as obvious an effect, although the 20 kPa PDGF + FGF-treated group had significantly longer vessels than the vascular endothelial growth factor-treated group. We have developed a novel, tunable hydrogel assay for the creation of lung explant vessel organoids which can be modulated to study the impact of specific environmental cues on vessel formation and maturation.

Control of fibrosis with enhanced safety via asymmetric inhibition of prolyl-tRNA synthetase 1.

Prolyl-tRNA synthetase 1 (PARS1) has attracted much interest in controlling pathologic accumulation of collagen containing high amounts of proline in fibrotic diseases. However, there are concerns about its catalytic inhibition for potential adverse effects on global protein synthesis. We developed a novel compound, DWN12088, whose safety was validated by clinical phase 1 studies, and therapeutic efficacy was shown in idiopathic pulmonary fibrosis model. Structural and kinetic analyses revealed that DWN12088 binds to catalytic site of each protomer of PARS1 dimer in an asymmetric mode with different affinity, resulting in decreased responsiveness at higher doses, thereby expanding safety window. The mutations disrupting PARS1 homodimerization restored the sensitivity to DWN12088, validating negative communication between PARS1 promoters for the DWN12088 binding. Thus, this work suggests that DWN12088, an asymmetric catalytic inhibitor of PARS1 as a novel therapeutic agent against fibrosis with enhanced safety.

Evaluating the Effect of Hydrogen Sulfide in the Idiopathic Pulmonary Fibrosis Model with a Fluorescent Probe.

Hydrogen sulfide (H2S), a gaseous signaling molecule, is involved in a wide range of physiological and pathological processes. H2S has been proven to play a beneficial role in lung diseases, and the relationship between perturbations in endogenous H2S synthesis and degree with idiopathic pulmonary fibrosis (IPF) has attacted increasing attention. However, the changes in endogenous lung H2S levels in the pathological progression of chronic pulmonary diseases remain unclear. To this end, we synthesized a fluorescent probe (Bcy-HS) for the selective imaging of H2S in living cells and mice. This probe was mainly used for in situ in vivo and cellular imaging as well as a systematic assessment of intrapulmonary H2S levels at different stages of IPF. In addition, we also discussed the potential of H2S supplementation in the treatment of pulmonary fibrotic diseases. Our results confirmed the key role of H2S in pulmonary fibrosis. In cellular and mice models of pulmonary fibrosis, intracellular H2S levels are reduced. However, the severity of oxidative damage and pulmonary fibrosis decreased after NaSH (H2S donor). Therefore, we concluded that increasing the H2S content in vivo may be a novel strategy for IPF treatment.

Artificial neural network identified the significant genes to distinguish Idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease that causes irreversible damage to lung tissue characterized by excessive deposition of extracellular matrix (ECM) and remodeling of lung parenchyma. The current diagnosis of IPF is complex and usually completed by a multidisciplinary team including clinicians, radiologists and pathologists they work together and make decision for an effective treatment, it is imperative to introduce novel practical methods for IPF diagnosis. This study provided a new diagnostic model of idiopathic pulmonary fibrosis based on machine learning. Six genes including CDH3, DIO2, ADAMTS14, HS6ST2, IL13RA2, and IGFL2 were identified based on the differentially expressed genes in IPF patients compare to healthy subjects through a random forest classifier with the existing gene expression databases. An artificial neural network model was constructed for IPF diagnosis based these genes, and this model was validated by the distinctive public datasets with a satisfactory diagnostic accuracy. These six genes identified were significant correlated with lung function, and among them, CDH3 and DIO2 were further determined to be significantly associated with the survival. Putting together, artificial neural network model identified the significant genes to distinguish idiopathic pulmonary fibrosis from healthy people and it is potential for molecular diagnosis of IPF.

Evaluation of Brilaroxazine (RP5063) in a Bleomycin-Induced Rodent Model of Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis pathology involves serotonin (5-HT), with an increased expression of 5-HT2A/2B/7 receptors in the lungs. This study tests the hypothesis that brilaroxazine (RP5063), an agent with a potent binding affinity for serotonin 5-HT1A/2A/2B/7 and dopamine D2/3/4 receptors, and moderate affinity for the 5-HT transporter, dosed at 15 mg twice daily (b.i.d.) shows efficacy as compared with placebo in a bleomycin (BLM)-induced model using Sprague Dawley rats. On Day 0, four groups received BLM-induction, and one received placebo. On Day 1, one group started on brilaroxazine (RPT). On Day 10, two groups started on brilaroxazine (RPI) and one continued on the vehicle (BLM). All interventions continued until Day 20. Compared with BLM, RPT and RPI sustained survival at 90.5% and 89.5%, respectively (P&lt;0.05) and maintained weight (P&lt;0.01). RPT normalized pulse pressure and cardiac output. It also lowered respiratory resistance, hydroxyproline, lung weight, bronchoalveolar lavage fluid cell counts, and total protein (P&lt;0.05). RPI decreased hydroxyproline and reduced cell counts (P&lt;0.01). Brilaroxazine lowered Ashcroft scores and Masson’s Trichome staining (P&lt;0.001). Both brilaroxazine groups reduced proinflammatory and fibrotic cytokines (P&lt;0.05). Brilaroxazine attenuated BLM-induced pulmonary fibrosis, inflammation, and extracellular deposition and improved cardiac and pulmonary functions in rodents.

The Restoring Effect of Human Umbilical Cord-Derived Mesenchymal Cell-Conditioned Medium (hMSC-CM) against Carbon Tetrachloride-Induced Pulmonary Fibrosis in Male Wistar Rats.

Pulmonary toxicity induced by CCl4, a model of idiopathic pulmonary fibrosis (IPF), leads to tissue remodeling and inflammation. Human umbilical cord mesenchymal cell-conditioned medium (hMSC-CM) is a potent anti-inflammatory, antioxidative, and antifibrotic agent. Forty male Wistar rats were assigned to the control (C), olive oil control (C.O) (hMSC-CM), control (C.Ms), fibrosis (fb), and fibrosis with hMSC-CM (f.Ms) treatment groups. The groups C, C.O, and C.Ms received PBS (200 µl), olive oil (1 ml/kg), and hMSC-CM (100 μg protein/kg), respectively. The fibrosis group was administered with only CCl4 (1 ml/kg). The last group, f.Ms was treated with CCl4 (1 ml/kg) and 100 μg protein/kg IV hMSC-CM. While the treatment with olive oil and CCl4 was performed for 2 days/week from the first week for 12 weeks, the treatment with PBS and hMSC-CM was carried out 2 days/week from week 4th to week 12th. The effect of the UC-MSC culture medium treatment on the lung was evaluated by assessing lysyl oxidase (LOX), tumor necrosis factor-alpha (TNF-α), and transforming growth factor-β1 (TGF-β1) genes, and proteins expression by real-time RCR and western blotting, respectively. Lysyl oxidase (LOX), tumor necrosis factor-alpha (TNF-α), transforming growth factor-b1 (TGF-β1), malondialdehyde (MDA), and oxidative stress levels were markedly higher in the fibrosis group than in the control groups (p ≤ 0.001). Additionally, glutathione (GSH) in the fibrosis group was markedly lower than those in the control groups (p ≤ 0.001). Fibrosis in the UC-MSC treatment group had milder histopathological injuries than in the fibrosis group. hMSC-MSC as a strong anti-inflammatory, antioxidative, and antifibrotic decreases the level of oxidative stress, proinflammatory cytokines, and MDA causing a restoring effect against CCl4-induced pulmonary fibrosis.

Human Lung Tissue Implanted on the Chick Chorioallantoic Membrane as a Novel In Vivo Model of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with no curative pharmacological treatment. Current preclinical models fail to accurately reproduce human pathophysiology and are therefore poor predictors of clinical outcomes. Here, we investigated whether the chick embryo chorioallantoic membrane (CAM) assay supports the implantation of xenografts derived from IPF lung tissue and primary IPF lung fibroblasts and can be used to evaluate the efficacy of antifibrotic drugs. We demonstrate that IPF xenografts maintain their integrity and are perfused with chick embryo blood. Size measurements indicate that the xenografts amplify on the CAM, and Ki67 and pro-collagen type I immunohistochemical staining highlight the presence of proliferative and functional cells in the xenografts. Moreover, the IPF phenotype and immune microenvironment of lung tissues are retained when cultivated on the CAM and the fibroblast xenografts mimic invasive IPF fibroblastic foci. Daily treatments of the xenografts with nintedanib and PBI-4050 significantly reduce their size, fibrosis-associated gene expression, and collagen deposition. Similar effects are found with GLPG1205 and fenofibric acid, two drugs that target the immune microenvironment. Our CAM-IPF model represents the first invivo model of IPF that uses human lung tissue. This rapid and cost-effective assay could become a valuable tool for predicting the efficacy of antifibrotic drug candidates for IPF.

Conditioned medium from a human adipose-derived stem cell line ameliorates inflammation and fibrosis in a lung experimental model of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis is a chronic, progressive parenchymal lung disease that results in fibrogenesis and the conditioned medium from adipose-derived mesenchymal stem cells (CM-ADSCs) has been shown to be efficacious in pulmonary fibrosis animal models. The aim of the present study is to evaluate the effect of CM-ADSCs on lung inflammation and fibrosis in a Bleomycin (BLM)-induced pulmonary fibrosis model. CM-ADSCs safety and toxicity were evaluated in Sprague Dawley rats and no adverse effects were observed. Six-week-old female C57BL/6J mice were employed in the BLM-induced pulmonary fibrosis model and were divided into four groups: Group 1 (Sham): animals were kept without BLM and treatment, Group 2 (Control): BLM with vehicle DMEM, Group 3: 10 μg/kg CM-ADSCs and Group 4: 100 μg/kg CM-ADSCs. Body weight, fibrosis and inflammation histological analyses, mRNA and protein pro-inflammatory cytokine, and total hydroxyproline content calculation were performed in all groups upon sacrifice. The 100 μg/kg CM-ADSCs showed a significant increase in mean body weight compared to Controls. CM-ADSCs doses resulted in the amelioration of fibrosis, as seen by Masson's Trichrome-staining, Ashcroft scoring, and Sirius red-staining. Compared to Controls, inflammation was also significantly reduced in CM-ADSCs-treated mice, with reduced F4/80 macrophage antigen staining, TNF-α mRNA and IL-6 and IL-10 protein levels. Total hydroxyproline content was found significantly reduced in both groups of CM-ADSCs-treated mice. Overall, our study shows that the CM-ADSCs is safe and efficient against pulmonary fibrosis, as it significantly reduced inflammation and fibrosis, with the larger dose of 100 μg/kg CM-ADSCs being the most efficient one.

PH-Triggered Aggregation of Gold Nanoparticles for Enhanced Labeling and Long-Term CT Imaging Tracking of Stem Cells in Pulmonary Fibrosis Treatment.

Gold nanoparticles (AuNPs) pose a great challenge in the development of nanotracers that can self-adaptively alter their properties in response to certain cellular environments for long-term stem cell tracking. Herein, pH-sensitive Au nanotracers (CPP-PSD@Au) are fabricated by sequential coupling of AuNPs with sulfonamide-based polymer (PSD) and cell-penetrating peptide (CPP), which can be efficiently internalized by mesenchymal stem cells (MSCs) and undergo pH-induced self-assembly in endosomes, facilitating long-term computed tomography (CT) imaging tracking MSCs in a murine model of idiopathic pulmonary fibrosis (IPF). Using the CPP-PSD@Au, the transplanted MSCs for the first time can be monitored with CT imaging for up to 35 days after transplantation into the lung of IPF mice, clearly elucidating the migration process of MSCs in vivo. Moreover, we preliminarily explored the mechanism of the CPP-PSD@Au labeled MSCs in the alleviation of IPF, including recovery of alveolar integrity, decrease of collagen deposition, as well as down-regulation of relevant cytokine level. This work facilitates our understanding of the behavior and effect of MSCs in the therapy of IPF, thereby providing an important insight into the stem cell-based treatment of lung diseases.

Examining lysyl oxidase-like modulation of collagen architecture in 3D spheroid models of idiopathic pulmonary fibrosis via second-harmonic generation microscopy.

.Significance: Idiopathic pulmonary fibrosis (IPF) patients have a poor prognosis with short lifespan following diagnosis as there are limited effective treatment options. Despite matrix stiffening being the hallmark of the disease there remains a lack of knowledge surrounding the underlying collagen alterations in the disease. Specifically, while increased collagen crosslinking has been implicated, the resulting effects on collagen macro/supramolecular changes have not been explored.Aim: We sought to determine if second-harmonic generation (SHG) microscopy could characterize differences in the collagen architecture in 3D spheroid models of IPF grown under different crosslinking modulation conditions (promotion and inhibition).Approach: We used SHG metrics based on the fiber morphology, relative SHG brightness, and macro/supramolecular structure by SHG polarization analyses to compare the structure of the IPF spheroids.Results: Comparison of the fiber morphology of the spheroids showed that the control group had the longest, straightest, and thickest fibers. The spheroids with crosslink enhancement and inhibition had the highest and lowest SHG conversion efficiencies, respectively, consistent with the resulting harmonophore density. SHG polarization analyses showed that the peptide pitch angle, alignment of collagen molecules, and overall chirality were altered upon crosslink modulation and were also consistent with reduced organization relative to the control group.Conclusions: While no single SHG signature is associated with crosslinking, we show that the suite of metrics used here is effective in delineating alterations across the collagen architecture sizescales. The results largely mirror those of human tissues and demonstrate that the combination of 3D spheroid models and SHG analysis is a powerful approach for hypothesis testing the roles of operative cellular and molecular factors in IPF.

Comparison of The Novel K Ca 3.1 Inhibitor SAN903 with Nintedanib and Pirfenidone In A Model of Idiopathic Pulmonary Fibrosis

Hypothesis Idiopathic pulmonary fibrosis (IPF) is a chronic and debilitating disease characterized by progressive lung fibrosis and declined function. IPF is a fatal condition with an expected survival time of 3-4 years, despite two medicines, nintedanib and pirfenidone, having been registered by the FDA/EMA. There is a clear need for more effective treatment options. KCa3.1 is a Ca2+-activated K+- channel expressed in fibroblasts/myofibroblasts, immune cells, and many epithelia/endothelia. The KCa3.1 channel is reported to control proliferation, migration, as well as cytokine release and collagen deposition in fibrosis models and inhibition of the KCa3.1 channel is hypothesized to ameliorate this inflammatory/fibrotic cascade. This study reports the testing of a novel KCa3.1 inhibitor, SAN903, compared to nintedanib and pirfenidone in a mouse model of IPF. Method C57BL/6J mice were given a single lung instillation of bleomycin (1.5 IU/kg) or saline (sham) and followed for 21 days. Groups of 2 × 8 animals (experiment lines 1 and 2) were dosed with SAN903 (30mg/kg, bid, po), nintedanib (60mg/kg, qd, po), pirfenidone (200mg/kg, bid, po), or vehicle (bid, po). Dose selection for nintedanib and pirfenidone were taken from the literature and adjusted to approach calculated human equivalent doses. Physical examination and body weights were recorded daily. At day 22, line 1 mice were sacrificed, and the lungs prepared for histochemical evaluation (H&E and MT staining). Line 2 mice were anesthetized and bronchoalveolar lavage fluid (BALF) collected for analysis. Statistical testing consisted of one-way/repeated measures ANOVA followed by Fisher´s LSD test with significance set at alpha P<0.05. Result Increase in histopathological score, biochemical parameters and cellular infiltration indicated robust and statistically significant inflammation/fibrosis in the vehicle group (p<0.0001 vs sham). No mortalities were reported during the study. SAN903 significantly (N=16) counteracted body weight decline caused by bleomycin, whereas nintedanib showed a trend towards improved outcomes and pirfenidone significantly worsened the decline. Histology revealed significant reductions of both inflammation and fibrosis in the SAN903 treated group (P=0.03), whereas nintedanib showed a numerical effect and pirfenidone trended towards impaired outcomes. The difference in effect between SAN903 and pirfenidone on fibrosis score was statistically significant (p=0.002). Analysis of BALF showed no significant effects on total or differential cell counts by any compound, whereas nintedanib significantly reduced TGF-1β (p=0.003) and SAN903 showed trend of inhibition (p=0.15). None of the compounds significantly decreased lung hydroxyproline contents. Conclusion: SAN903 reduced lung inflammation and fibrosis in the mouse bleomycin-model of lung fibrosis. The effects of SAN903 on lung pathology seem superior to the two standard-of-care medicines for IPF.

Discovery of a Potent and Selective Covalent Inhibitor and Activity-Based Probe for the Deubiquitylating Enzyme UCHL1, with Antifibrotic Activity.

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a deubiquitylating enzyme that is proposed as a potential therapeutic target in neurodegeneration, cancer, and liver and lung fibrosis. Herein we report the discovery of the most potent and selective UCHL1 probe (IMP-1710) to date based on a covalent inhibitor scaffold and apply this probe to identify and quantify target proteins in intact human cells. IMP-1710 stereoselectively labels the catalytic cysteine of UCHL1 at low nanomolar concentration in cells. We further demonstrate that potent and selective UCHL1 inhibitors block pro-fibrotic responses in a cellular model of idiopathic pulmonary fibrosis, supporting the potential of UCHL1 as a potential therapeutic target in fibrotic diseases.