Bleomycin In Rats Research Articles

Combination of losartan with pirfenidone: a protective anti-fibrotic against pulmonary fibrosis induced by bleomycin in rats

Pirfenidone (PFD), one acceptable medication for treating idiopathic pulmonary fibrosis (IPF), is not well tolerated by patients at full doses. Hence, employing of some approaches such as combination therapy may be applicable for increasing therapeutic efficacy of PFD. Losartan (LOS), an angiotensin II receptor antagonist, could be a suitable candidate for combination therapy because of its stabilizing effect on the pulmonary function of IPF patients. Therefore, this study aimed to investigate the effects of LOS in combination with PFD on bleomycin (BLM)-induced lung fibrosis in rats. BLM-exposed rats were treated with LOS alone or in combination with PFD. The edema, pathological changes, level of transforming growth factor-β (TGF-β1), collagen content, and oxidative stress parameters were assessed in the lung tissues. Following BLM exposure, the inflammatory response, collagen levels, and antioxidant markers in rat lung tissues were significantly improved by PFD, and these effects were improved by combination with LOS. The findings of this in vivo study suggest that the combined administration of PFD and LOS may provide more potent protection against IPF than single therapy through boosting its anti-inflammatory, anti-fibrotic, and anti-oxidant effects. These results hold promise in developing a more effective therapeutic strategy for treating of lung fibrosis.

Insights into Disease Progression of Translational Preclinical Rat Model of Interstitial Pulmonary Fibrosis through Endpoint Analysis.

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease characterized by the relentless deposition of extracellular matrix (ECM), causing lung distortions and dysfunction. Animal models of human IPF can provide great insight into the mechanistic pathways underlying disease progression and a means for evaluating novel therapeutic approaches. In this study, we describe the effect of bleomycin concentration on disease progression in the classical rat bleomycin model. In a dose-response study (1.5, 2, 2.5 U/kg i.t), we characterized lung fibrosis at day 14 after bleomycin challenge using endpoints including clinical signs, inflammatory cell infiltration, collagen content, and bronchoalveolar lavage fluid-soluble profibrotic mediators. Furthermore, we investigated fibrotic disease progression after 2 U/kg i.t. bleomycin administration at days 3, 7, and 14 by quantifying the expression of clinically relevant signaling molecules and pathways, epithelial mesenchymal transition (EMT) biomarkers, ECM components, and histopathology of the lung. A single bleomycin challenge resulted in a progressive fibrotic response in rat lung tissue over 14 days based on lung collagen content, histopathological changes, and modified Ashcroft score. The early fibrogenesis phase (days 3 to 7) is associated with an increase in profibrotic mediators including TGFβ1, IL6, TNFα, IL1β, CINC1, WISP1, VEGF, and TIMP1. In the mid and late fibrotic stages, the TGFβ/Smad and PDGF/AKT signaling pathways are involved, and clinically relevant proteins targeting galectin-3, LPA1, transglutaminase-2, and lysyl oxidase 2 are upregulated on days 7 and 14. Between days 7 and 14, the expressions of vimentin and α-SMA proteins increase, which is a sign of EMT activation. We confirmed ECM formation by increased expressions of procollagen-1Aα, procollagen-3Aα, fibronectin, and CTGF in the lung on days 7 and 14. Our data provide insights on a complex network of several soluble mediators, clinically relevant signaling pathways, and target proteins that contribute to drive the progressive fibrotic phenotype from the early to late phase (active) in the rat bleomycin model. The framework of endpoints of our study highlights the translational value for pharmacological interventions and mechanistic studies using this model.

Modified Qing-Zao-Jiu-Fei decoction attenuated pulmonary fibrosis induced by bleomycin in rats via modulating Nrf2/NF-κB and MAPKs pathways

BackgroundQing-Zao-Jiu-Fei Decoction (QZJFD) is a famous herbal formula commonly prescribed for the treatment of lung-related diseases in the ancient and modern times. Trichosanthis Fructus (TF) and Fritillariae Thunbergii Bulbus (FTB) are widely used for treatment of cough and pulmonary disease. In order to identify a more effective formula for treatment of pulmonary fibrosis, we intend to add TF and FTB in QZJFD to form a modified QZJFD (MQZJFD). In this study, we aims to explore MQZJFD as an innovative therapeutic agent for pulmonary fibrosis using bleomycin (BLM)-treated rats and to unravel the underlying molecular mechanisms.MethodsBLM was given to SD rats by intra-tracheal administration of a single dose of BLM (5 mg/kg). QZJFD (3 g/kg) and MQZJFD (1, 2 and 4 g/kg) was given intragastrically daily to rats for 14 days (from day 15 to 28) after BLM administration for 14 consecutive days.ResultsMQZJFD was found to contain 0.29% of amygdalin, 0.020% of lutin, 0.077% of glycyrrhizic acid and 0.047% of chlorogenic acid. BLM treatment could induce collagen deposition in the lung tissues of rats, indicating that the pulmonary fibrosis rat model had been successfully established. MQZJFD have better effects than the original QZJFD in reducing the pulmonary structure damage and collagen deposition of rat lung fibrosis induced by BLM. MQZJFD could reduce the hydroxyproline content in lung tissues of BLM-treated rats. The biomarkers of fibrosis such as matrix metalloproteinase 9 (MMP9), collagen I and α-smooth muscle actin (α-SMA) were remarkably reduced after treatment with MQZJFD. MQZJFD also have anti-oxidant stress effects by inhibiting the level of malondialdehyde (MDA), but enhancing the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the level of glutathione (GSH) in the lung tissues of BLM-treated rats. Moreover, the MQZJFD markedly suppressed the over expressions of p-p65/p65 and p-IκBα/IκBα, but upregulated the Nrf2. MQZJFD also suppressed the protein expressions of p-ERK1/2/ERK1/2, p-p38/p38 and p-JNK/JNK in the lung tissues of BLM-treated rats.ConclusionsMQZJFD could improve the pulmonary fibrosis induced by BLM in rats via inhibiting the fibrosis and oxidative stress via suppressing the activation of NF-κB/Nrf2 and MAPKs pathways.

Protective Effect of Tunisian Red Seaweed (Corallina officinalis) Against Bleomycin-Induced Pulmonary Fibrosis and Oxidative Stress in Rats.

Idiopathic pulmonary fibrosis is a chronic and progressive respiratory disease whose diagnosis and physiopathogenesis are still poorly understood and for which, until recently, there were no effective treatments. Over the past few decades, many studies have demonstrated that marine macroalgae such as red seaweeds are potential alternative sources of useful bioactive compounds possessing various physiological and biological activities. The present study was aimed to investigate the effect of Corallina officinalis aqueous extract (COAE) against bleomycin (BLM)-induced lung fibrosis in rat. Thus, Wistar rats were divided into 4 groups of 10 each: control, BLM (2mg/kg), BLM/COAE-150mg/kg and BLM/COAE-300mg/kg once a day for 21 days. Obtained results showed that COAE is rich in phenolic compounds and exhibited relatively high antioxidant activity. COAE might significantly reduce the damage caused by BLM by rewarding the decline in weight and pulmonary index in rats given only BLM. Moreover, lungs, liver and kidneys lipid peroxidation, and sulfhydryl group levels were reversed significantly in a dose-dependent manner in the COAE-treated groups. BLM decreased superoxide dismutase (SOD) and catalase (CAT) activities, while COAE administration increased the antioxidant enzyme activities. Histopathologically, COAE attenuates the severity of the inflammatory lungs state caused by instillation of BLM in rats. These findings suggest that COAE can be a potential therapeutic candidate against BLM-induced lung fibrosis.

Characterization of acute lung injury in the bleomycin rat model.

The aim of this study was to describe and characterize the pathophysiological changes occurring during the early inflammatory phase (first 3 days) in the rat bleomycin model of lung injury preceding the development of fibrosis. Further, we wanted to understand the kinetics and factors contributing to bleomycin-induced acute lung injury (ALI) and provide a robust, reliable and reproducible framework of features of ALI readouts to assess effects of therapeutics on bleomycin-induced ALI in rats. We induced ALI in rats with intratracheal (i.t.) installation of bleomycin. The animals were sacrificed on predetermined time points, that is, Day 0, 1, 2, and 3 post the bleomycin challenge. We analyzed bronchoalveolar lavage fluid (BALF) and lung tissue to establish and assess relevant experimental features of ALI. We demonstrated that bleomycin induced key features of experimental ALI including a profound increase in neutrophils in BALF (50-60%), pulmonary edema, and lung pathology on Day 3 after challenge. Furthermore, we showed that TGF-β1, IL-1β, TNF-α, IL-6, CINC-1, TIMP-1, and WISP-1 were induced by studying their kinetic profile during the first 3 days after bleomycin injury consistent with their known role ALI. We also confirmed that detectable fibrogenesis occurs at the earliest on Day 3 after injury based on collagen content, along with changes in the TGF-β/Smad signaling pathway and increased expression of Galectin-3, Vimentin, and Fibronectin in lung homogenate. Our report presents robust features and contributing mediators/factors to the pathology of bleomycin-induced ALI in rats on Day 3. The kinetic data provide insights on the progression of ALI and a detailed understanding of early events before actual fibrosis development. This set of experimental endpoints is very appropriate and invaluable for efficacy testing of potential novel therapeutic treatments (single or combined) in ALI and understanding their mechanism of action.

Enhanced secretion of hepatocyte growth factor in human umbilical cord mesenchymal stem cells ameliorates pulmonary fibrosis induced by bleomycin in rats.

Umbilical cord mesenchymal stem cells (UCMSCs) are a reportedly promising choice in the treatment of irreversible pulmonary fibrosis and lethal interstitial lung disease with limited drug treatment options. In this study, we investigated the therapeutic efficacy of UCMSCs overexpressing hepatocyte growth factor (HGF), which is considered one of the main anti-fibrotic factors secreted by MSCs. Adenovirus vector carrying the HGF gene was transfected into UCMSCs to produce HGF-modified UCMSCs (HGF-UCMSCs). Transfection promoted the proliferation of UCMSCs and did not change the morphology, and differentiation ability, or biomarkers. Rats were injected with HGF-UCMSCs on days 7 and 11 after intratracheal administration of bleomycin (10mg/kg). We performed an analysis of histopathology and lung function to evaluate the anti-fibrotic effect. The results showed that HGF-UCMSCs decreased the Ashcroft scores in hematoxylin and eosin-stained sections, the percentage positive area in Masson trichrome-stained sections, and the hydroxyproline level in lungs. Forced expiratory volume in the first 300m/forced vital capacity was also improved by HGF-UCMSCs. To explore the possible therapeutic mechanism of HGF-UCMSCs, we detected inflammatory factors in the lungs and performed mRNA sequencing in UCMSCs and HGF-UCMSCs. The data indicated that inhibition of interleukin-17 in the lung may be related to the anti-fibrosis of HGF-UCMSCs, and overexpressed HGF probably played a primary role in the treatment. Collectively, our study findings suggested that the overexpression of HGF may improve the anti-fibrotic effect of UCMSCs through directly or indirectly interacting with interleukin-17-producing cells in fibrotic lungs.

Preparation and physicochemical characterization of N-succinyl chitosan-coated liposomes for oral delivery of grape seed extract and evaluation of its effect on pulmonary fibrosis induced by bleomycin in rats.

This study aimed to develop an oral succinyl chitosan-coated liposomal formulation containing grape seed extract and assess its therapeutic efficacy in rats with bleomycin-induced pulmonary fibrosis. N-succinyl chitosan was synthesized, and the liposomal formulations were prepared and characterized regarding phenolic content assay and morphology. Size, zeta potential, in vitro drug release, and stability. Pulmonary fibrosis was induced by intratracheal bleomycin injection, and hydroxyproline measurements, lung weight, animal body weight, as well as histopathological studies were performed. Succinyl chitosan increases the physical stability of the formulation, especially in acidic conditions. Drug release studies revealed that 66.27% of the loaded drug was released from CF2 in an acidic medium in 2 hr, but 92.31% of the drug was released in 8 hr in a pH=7 medium. An in vivo study demonstrated that rats exposed to bleomycin significantly lost weight, while those treated with CF2 (400 mg/kg) partially regained weight. Bleomycin treatment increased the mean lung weight and the amount of hydroxyproline in the lungs; these values were significantly decreased in the group treated with 400 mg/kg CF2 (P<0.05). Histopathological examination confirmed that treatment with 400 mg/kg CF2 improved lung fibrosis. In rats, oral administration of N-succinyl chitosan-coated liposomes containing grape seed extract at the 400 mg/kg dose ameliorates bleomycin-induced pulmonary fibrosis.

PEDF is an antifibrosis factor that inhibits the activation of fibroblasts in a bleomycin-induced pulmonary fibrosis rat model

BackgroundIdiopathic pulmonary fibrosis (IPF) is a highly heterogeneous and fatal lung disease. In addition to dense fibrous tissue, abnormal angiogenesis is also an important feature of IPF. Pigment epithelium-derived factor (PEDF) is an angiogenesis inhibitor and a potential anti-fibrous factor. The purpose of this experiment is to observe the effect of PEDF on bleomycin (BLM)-induced pulmonary fibrosis in rats.MethodsIn vivo, pathological examination and detection of related factors were performed on pulmonary fibrosis induced by BLM in rats, and the temporal and spatial distribution of PEDF was investigated. Furthermore, lung gene delivery (PEDF-adeno-associated virus) was performed to investigate the effect of PEDF on pulmonary fibrosis. In vitro, lentiviral vectors were used to construct PEDF over-expression or knock out primary rat lung (PRL) fibroblasts. The effect of PEDF on fibroblast activation under TGF-β1 stimulation was evaluated, and the activation of TGF-β1/smad pathway and PPAR-γ expression (in the presence or absence of PPAR-γ inhibitors) were analyzed.ResultsIn vivo results showed that PEDF expression decreased during the inflammatory phase and increased during the fibrotic phase. PEDF could inhibit the progression of pulmonary fibrosis in rats. In vitro results showed that PEDF could effectively inhibit TGF-β1-stimulated fibroblast activation and reduce the production of α-SMA and collagen-I. PEDF could inhibit the TGF-β1/smad pathway by up-regulating the activity of PPAR-γ.ConclusionsPEDF can act as an anti-fibrotic factor, inhibit fibroblast activation by upregulating PPAR-γ activity and reduce BLM-induced pulmonary fibrosis in rats.

Icariin attenuates bleomycin-induced pulmonary fibrosis by targeting Hippo/YAP pathway

Pulmonary fibrosis is a manifestation of the progression of interstitial pulmonary disease. Icariin (ICA) has been found to exhibit protective effects on multiple chronic diseases like diabetes, liver, heart, and renal fibrosis. Here, a systemic pharmacological study was designed to investigate whether ICA treatment alleviates bleomycin (BLM)-induced pulmonary fibrosis. The rat pulmonary fibrosis model was constructed by non-invasive endotracheal intubation instillation of BLM to observe the intervention effects of ICA on pulmonary fibrosis in the whole process of inflammation and fibrosis. ICA reduced the collagen deposition and inflammation induced by BLM in rat. The comparative RNA-sequencing was conducted to analyze the lung gene expression profiles in rat. KEGG analysis indicated that most of the genes were enriched in Hippo pathway, NF-κB pathway, and B-cell receptor signaling pathway, etc. Immunohistochemistry staining showed that the expression of YAP was significantly elevated in the model group and decreased in the ICA treatment group. Taken together, the anti-fibrotic effect of ICA appears to be mediated by its inhibitory of YAP, which is the core transcriptional regulator of Hippo pathway.

The Protective Effects of Nebulized Nano-Curcumin Against Bleomycin-Induced Pulmonary Fibrosis in Rats

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive fatal disease affecting the lung, and currently there is no efficient therapy for this condition. Curcumin, as a natural anti-inflammatory and antioxidant agent, could repress the pulmonary fibrosis (PF) caused by Bleomycin (BLM). Objectives: The aim of the research was to evaluate the protective activity of a nano-formulation of curcumin administered by inhalation on BLM-induced PF in rats. Methods: Eighty rats were randomly divided into eight experimental groups. Group one (control) that received saline intratracheally (IT) and subjected to vehicle inhalation. Group two to eight each received a single dose of BLM (5 IU/kg, IT) along with vehicle inhalation, oral prednisolone, oral curcumin, curcumin inhalation, and nano-curcumin inhalation with the doses of 50, 100, and 200 µg/kg, respectively. In the control and other groups, BLM was injected intratracheally on the first day of the experiment. In the treatment groups, curcumin suspension was prepared in distilled water and applied through nebulization for 21 consecutive days after BLM intratracheal administration. Then the rats were euthanized, and inflammatory cytokines (TNF-α, TGF-β, PDGF), hydroxyproline, and IL-10 (as a protective cytokine) were measured. Also, lung histopathological features were examined. Results: The synthesized nano-formulation reduced the overall hydroxyproline content of lungs in BLM-treated rats (P &lt; 0.002). In addition, TNF-α, TGF-β, and PDGF levels significantly increased in the lungs of BLM-instilled rats (P &lt; 0.001). However, the nano-formulation of curcumin (200 µg/kg) significantly decreased the levels of these inflammatory cytokines (P &lt; 0.001) and increased IL-10 level (P = 0.0144) compared with the control group. Conclusions: The nebulization of nano-curcumin is suggested as a novel approach for the treatment of PF induced by BLM in rats. Our findings revealed that the inhalation (as a safe local drug delivery system approach) of the nano-curcumin at a dose of 200 µg/kg (formulated by cyclodextrin) could effectively protect the lung against PF.

D-Limonene Is a Potential Monoterpene to Inhibit PI3K/Akt/IKK-α/NF-κB p65 Signaling Pathway in Coronavirus Disease 2019 Pulmonary Fibrosis.

At the time of the prevalence of coronavirus disease 2019 (COVID-19), pulmonary fibrosis (PF) related to COVID-19 has become the main sequela. However, the mechanism of PF related to COVID (COVID-PF) is unknown. This study aimed to explore the key targets in the development of COVID-PF and the mechanism of d-limonene in the COVID-PF treatment. The differentially expressed genes of COVID-PF were downloaded from the GeneCards database, and their pathways were analyzed. d-Limonene was molecularly docked with related proteins to screen its pharmacological targets, and a rat lung fibrosis model was established to verify d-limonene's effect on COVID-PF-related targets. The results showed that the imbalance between collagen breakdown and metabolism, inflammatory response, and angiogenesis are the core processes of COVID-PF; and PI3K/AKT signaling pathways are the key targets of the treatment of COVID-PF. The ability of d-limonene to protect against PF induced by bleomycin in rats was reported. The mechanism is related to the binding of PI3K and NF-κB p65, and the inhibition of PI3K/Akt/IKK-α/NF-κB p65 signaling pathway expression and phosphorylation. These results confirmed the relationship between the PI3K–Akt signaling pathway and COVID-PF, showing that d-limonene has a potential therapeutic value for COVID-PF.

Bleomycin induces fibrotic transformation of bone marrow stromal cells to treat height loss of intervertebral disc through the TGF\u03b2R1/Smad2/3 pathway

BackgroundLower back pain is often accredited to loss of intervertebral disc (IVD) height and compromised spine stability as a result of intervertebral disc degeneration (IVDD). We aim to locally use bleomycin to induce the fibrotic transformation of bone marrow stromal cells (BMSCs) as a means to induce reparative fibrosis to slow down the height loss.MethodsIVDs from patients were gathered for histological examination. The expression of the transforming growth factor beta 1 (TGF-β) signaling pathway was determined by qPCR and western blotting. Nucleus pulposus (NP) cells, annulus fibrosus (AF) cells, and the rats’ bone marrow stromal cells (BMSC) were cultured and their responsiveness to bleomycin was evaluated by Cell Counting Kit-8, comet assay, transwell migration, and wound healing assays. Rat IVDD models were created by puncture and rescued by bleomycin injection, and the effectiveness was evaluated by images (X-ray and MRI) and atomic force microscope.ResultsHistological examination showed increased levels of pro-fibrotic markers in IVDD tissues from patients. AF cells and BMSC cells were induced to adopt a pro-fibrotic phenotype with increased expression fibrotic markers Col1a1, Col3a1, and FSP1. The pro-fibrotic effect of bleomycin on AF cells and BMSCs was in part due to the activation of the TGFβ-TGFβR1-SMAD2/3 signaling pathway. Pharmacological inhibition or gene knock-down of TGFβR1 could mitigate the pro-fibrotic effects.ConclusionLocally, injection of bleomycin in rats’ IVD induced rapid fibrosis and maintained its height through the TGFβ-TGFβR1-SMAD2/3 signaling pathway.

Dysregulation of BMP9/BMPR2/SMAD signalling pathway contributes to pulmonary fibrosis and pulmonary hypertension induced by bleomycin in rats

Pulmonary hypertension related to pulmonary fibrosis is classed as WHO Group III, one of the most common groups which lacks effective treatment options. In this study, we aimed to uncover the underlying mechanisms, particularly the involvement of the BMP9/BMPR2/SMAD signalling pathway, in this subtype of pulmonary hypertension. Male Sprague Dawley rats were used to establish a model of pulmonary hypertension with pulmonary fibrosis, induced by bleomycin. Haemodynamic and lung functions were measured, along with histological and immunohistochemical examinations. Primary cultures of rat pulmonary microvascular endothelial cells (PMVECs) were analysed with western blots, apoptosis assays and immunohistochemistry. Early (7 days) after bleomycin treatment of rats, pulmonary arterial thickening and severe loss of pulmonary arterial endothelium were observed, followed (14 days) by increased right ventricular systolic pressure and right ventricular hypertrophy. Marked down-regulation of the BMP9/BMPR2/SMAD signalling pathway was markedly down-regulated in lung tissues from bleomycin-treated rats (throughout the 7- to 35-day treatment period) and bleomycin-treated rat PMVECs, along with excessive cell apoptosis and loss of pulmonary arterial endothelium. Treatment with recombinant human bone morphogenetic protein 9 (rhBMP9) attenuated these aspects of bleomycin-induced pulmonary hypertension, by restoring disrupted BMP9/BMPR2/SMAD signalling. In bleomycin-treated rats, early and persisting suppression of the BMP9/BMPR2/SMAD signalling pathway triggered severe loss of pulmonary arterial endothelium and subsequent pulmonary arterial vascular remodelling, contributing to the development of pulmonary hypertension. Therapeutic approaches reinforcing BMP9/BMPR2/SMAD signalling might be ideal strategies for this subtype of pulmonary hypertension. This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.1/issuetoc.

Novel uses of ultrasound in various lung diseases

Conventional ultrasound (CUS) has been used in the chest to visualize pleural and pericardial fluid collections, diaphragm motion, and blood flow through the heart for anatomic definition and determining pressure gradients across valves. CUS is NOT suitable for imaging lung parenchyma, because of ultrasound multiple scattering (USMS) from millions of air-filled alveoli providing air-tissue interfaces. We used USMS to characterize lung tissue. We measured scattered mean free path (SMFP), the average distance between two scattering events, related to the amount of air present in lung parenchyma, and backscatter frequency shift (BFS), a measure of ultrasound attenuation. Using a rat bleomycin pulmonary fibrosis model, we showed that SMFP was longer in 18 rat lungs with pulmonary fibrosis, compared to 6 normals, and correlated with severity of lung fibrosis assessed by ex vivo CT scan and inflation-fixed histology. SMFP was significantly longer in 6 rat lungs made edematous by ischemia-reperfusion injury with significantly lower BFS. These data support using USMS to characterize severity of pulmonary fibrosis and pulmonary edema due to heart failure, and pneumonia and congestion in humans. In seven large animal lungs, determining the absence of USMS allows localization of pulmonary nodules for minimally invasive surgery. USMS provides many clinical opportunities.

Inhibition of mast cells: a novel mechanism by which nintedanib may elicit anti-fibrotic effects

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease which presents a grave prognosis for diagnosed patients. Nintedanib (a triple tyrosine kinase inhibitor) and pirfenidone (unclear mechanism of...

Biochanin-A ameliorates pulmonary fibrosis by suppressing the TGF-β mediated EMT, myofibroblasts differentiation and collagen deposition in in vitro and in vivo systems

Background: Idiopathic Pulmonary Fibrosis (IPF) is a progressive inflammatory disorder driven by a fibrotic cascade of events such as epithelial to mesenchymal transition, extracellular matrix production and collagen formation in the lungs in a sequential manner. IPF incidences were raising rapidly across the world. FDA approved pirfenidone and nintedanib (tyrosine kinase inhibitors) are being used as a first-line treatment drugs for IPF, however, neither the quality of life nor survival rates have been improved because of patient noncompliance due to multiple side effects. Thus, the development of novel therapeutic approaches targeting TGF-β mediated cascade of fibrotic events is urgently needed to improve the survival of the patients suffering from devastating disease.Purpose: The aim of this study was to investigate and validate the anti-fibrotic properties of Biochanin-A (isoflavone) against TGF-β mediated fibrosis in in vitro, ex vivo, in vivo models and to determine the molecular mechanisms that mediate these anti-fibrotic effects.Methods: The therapeutic activity of BCA was determined in in vitro/ex vivo models. Cells were pre-treated with BCA and incubated in presence or absence of recombinant-TGF-β to stimulate the fibrotic cascade of events. Pulmonary fibrosis was developed by intratracheal administration of bleomycin in rats. BCA treatment was given for 14 days from post bleomycin instillation and then various investigations (collagen content, fibrosis gene/protein expression and histopathological changes) were performed to assess the anti-fibrotic activity of BCA.Results:In vitro/ex vivo (Primary normal, IPF cell line and primary IPF cells/ Precision cut mouse lung slices) experiments revealed that, BCA treatment significantly (p < 0.001) reduced the expression of TGF-β modulated fibrotic genes/protein expressions (including their functions) which are involved in the cascade of fibrotic events. BCA treatment significantly (p < 0.01) reduced the bleomycin-induced inflammatory cell-infiltration, inflammatory markers expression, collagen deposition and expression of fibrotic markers in lung tissues equivalent or better than pirfenidone treatment. In addition, BCA treatment significantly (p < 0.001) attenuated the TGF-β1/BLM-mediated increase of TGF-β/Smad2/3 phosphorylation and resulted in the reduction of pathological abnormalities in lung tissues determined by histopathology observations.Conclusion: Collectively, BCA treatment demonstrated the remarkable therapeutic effects on TGF-β/BLM mediated pulmonary fibrosis using IPF cells and rodent models. This current study may offer a novel treatment approach to halt and may be even rescue the devastating lung scarring of IPF.

Alterations in Thiol/disulphide Homeostasis of Acute Lung Toxicity of Bleomycin in Rats

Alterations in Thiol/disulphide Homeostasis of Acute Lung Toxicity of Bleomycin in Rats

Pirfenidone suppresses bleomycin-induced pulmonary fibrosis and periostin expression in rats.

The aim of the present study was to investigate the effect of pirfenidone on bleomycin-induced lung fibrosis in rats, in order to elucidate the underlying mechanism of periostin-induced fibrosis. The lung fibrosis model was constructed using a single intratracheal instillation of bleomycin in rats. The normal rats without bleomycin administration were used as controls (n=24). Bleomycin-treated rats were randomized into the model (M) or pirfenidone (P) group (n=24 per group). Rats were sacrificed on days 7, 14 and 28 following treatment. Hematoxylin-eosin and Masson's trichrome staining were performed to analyze pulmonary alveolitis and fibrosis. Periostin location was detected by immunohistochemistry. Hydroxyproline content, and expression of periostin and transforming growth factor (TGF)-β1 were detected by ELISA, reverse transcription-quantitative polymerase chain reaction or western blotting. Correlation of periostin expression with hydroxyproline and TGF-β1 content was also analyzed. Histological findings demonstrated that pirfenidone significantly inhibited bleomycin-induced lung fibrosis and reduced the hydroxyproline content on day 14 and day 28 compared with the model group (P<0.05 or P<0.01). Furthermore, the bleomycin-induced increased protein expression of periostin and TGF-β1 was also significantly suppressed by pirfenidone on days 14 (P<0.01) and 28 (data not shown). Furthermore, periostin expression was significantly correlated with hydroxyproline and TGF-β1 content, and fibrosis score (P<0.001). The present findings suggest that the antifibrotic effect of pirfenidone may be achieved by suppression of periostin and TGF-β1 expression in rat pulmonary fibrogenesis.

Protective effect of hydroxysafflor yellow A on bleomycin- induced pulmonary inflammation and fibrosis in rats.

To observe the effect of hydroxysafflor yellow A (HSYA), an active ingredient of a traditional Chinese herbal medicine Carthamus tinctorius L., on lung inflflammation and pulmonary fibrosis induced by bleomycin (BLM) in rats. Animals were divided into 6 groups including normal group, model group, three HSYA groups and dexamethasone (DXM) group. Three doses of HSYA (35.6, 53.3, and 80.0 mg•kg-1•day-1) were intraperitoneally (i.p.) injected in rats for 3 weeks after BLM administration and DXM was used as the positive control (n=8 or 12). Arterial blood gas was assayed and morphological changes were observed. Lung mRNA expressions of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and some cytokines in lung tissue were detected by real-time polymerase chain reaction. Nuclear factor-κB p65 or α-smooth muscle actin (α-SMA) protein distribution in rat lung tissue was observed by immunohistochemistry. On the 7th day after BLM administration, lung tissue showed serious inflammation. Treatment with HSYA or DXM ameliorated lung inflammation. After treatment with HSYA or DXM, oxygen partial pressure (PaO2) increased (HSYA 80.0 mg•kg-1, P<0.01) and CO2 partial pressure (PaCO2) decreased (HSYA 53.3, 80.0 mg•kg-1, P<0.05). Moreover, the mRNA expression of TNF-α, IL-1β, and IL-6; and the number of NF-κB p65 positive cells was lower in HSYA 53.3 and 80.0 mg•kg-1 groups than those in the model group (all P<0.05). Twenty-one days after BLM administration, HSYA or DXM treatment ameliorated fibrosis, increased PaO2 (HSYA 53.3, 80.0 mg•kg-1, P<0.01), and decreased PaCO2 (53.3 and 80.0 mg•kg-1, P<0.05). Further, the mRNA expression of TGF-β1, α-SMA, and collagen I as well as the number of α-SMA positive cells increased in the model group and HSYA can attenuate these changes (53.3, 80.0 mg•kg-1, P<0.05). Hematoxylin and eosin and Masson's trichrome staining indicated that the fibrosis and collagen deposition were ameliorated in HSYA groups (53.3, 80.0 mg•kg-1, P<0.05). HSYA could alleviate acute lung inflflammation and chronic pulmonary fibrosis induced by BLM in rats.

Grape seed extract ameliorates bleomycin-induced mouse pulmonary fibrosis

Pulmonary fibrosis is common in a variety of inflammatory lung diseases, such as interstitial pneumonia, chronic obstructive pulmonary disease, and silicosis. There is currently no effective clinical drug treatment. It has been reported that grape seed extracts (GSE) has extensive pharmacological effects with minimal toxicity. Although it has been found that GSE can improve the lung collagen deposition and fibrosis pathology induced by bleomycin in rat, its effects on pulmonary function, inflammation, growth factors, matrix metalloproteinases and epithelial-mesenchymal transition remain to be researched. In the present study, we studied whether GSE provided protection against bleomycin (BLM)-induced mouse pulmonary fibrosis. ICR strain mice were treated with BLM in order to establish pulmonary fibrosis models. GSE was given daily via intragastric administration for three weeks starting at one day after intratracheal instillation. GSE at 50 or 100mg/kg significantly reduced BLM-induced inflammatory cells infiltration, proinflammatory factor protein expression, and hydroxyproline in lung tissues, and improved pulmonary function in mice. Additionally, treatment with GSE also significantly impaired BLM-induced increases in lung fibrotic marker expression (collagen type I alpha 1 and fibronectin 1) and decreases in an anti-fibrotic marker (E-cadherin). Further investigation indicated that the possible molecular targets of GSE are matrix metalloproteinases-9 (MMP-9) and TGF-β1, given that treatment with GSE significantly prevented BLM-induced increases in MMP-9 and TGF-β1 expression in the lungs. Together, these results suggest that supplementation with GSE may improve the quality of life of lung fibrosis patients by inhibiting MMP-9 and TGF-β1 expression in the lungs.