Atractylodin Suppresses TGF-β-Mediated Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells and Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is characterized by fibrotic change in alveolar epithelial cells and leads to the irreversible deterioration of pulmonary function. Transforming growth factor-beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in type 2 lung epithelial cells contributes to excessive collagen deposition and plays an important role in IPF. Atractylodin (ATL) is a kind of herbal medicine that has been proven to protect intestinal inflammation and attenuate acute lung injury. Our study aimed to determine whether EMT played a crucial role in the pathogenesis of pulmonary fibrosis and whether EMT can be utilized as a therapeutic target by ATL treatment to mitigate IPF. To address this topic, we took two steps to investigate: 1. Utilization of anin vitro EMT model by treating alveolar epithelial cells (A549 cells) with TGF-β1 followed by ATL treatment for elucidating the underlying pathways, including Smad2/3 hyperphosphorylation, mitogen-activated protein kinase (MAPK) pathway overexpression, Snail and Slug upregulation, and loss of E-cadherin. Utilization of an in vivo lung injury model by treating bleomycin on mice followed by ATL treatment to demonstrate the therapeutic effectiveness, such as, less collagen deposition and lower E-cadherin expression. In conclusion, ATL attenuates TGF-β1-induced EMT in A549 cells and bleomycin-induced pulmonary fibrosis in mice.     

霉酚酸酯对小鼠肺纤维化组织中转化生长因子-β1表达的影响

目的探讨霉酚酸酯(mycophenolate mofetil,MMF)对博莱霉素(bleomycin,BLM)诱导的小鼠肺纤维化组织中转化生长因子-β1(transforming growth factor-β1,TGF-β1)表达的影响。方法将C57BL/6小鼠随机分为6组:正常对照组、MMF对照组、BLM模型组、MMF低(20 mg/kg)、中(60 mg/kg)、高(100 mg/kg)剂量干预组,每组6只。BLM模型组和MMF 3个干预组经气管注入BLM(6 mg/kg),正常对照组和MMF对照组注入等量无菌生理盐水;次日按MMF对照组和MMF干预低、中、高剂量组小鼠体重计算MMF给药量,灌胃小鼠,每日1次,连续14 d,正常对照组和BLM模型组用等体积的双蒸水灌胃。灌胃第16天采集小鼠肺脏标本,经HE、Masson染色,从组织形态学上观察肺组织纤维化情况并进行Aschcroft评分;RT-PCR法检测小鼠肺组织中TGF-β1基因mRNA的转录水平;Western blot法检测小鼠肺组织中TGF-β1蛋白的表达水平。结果 BLM诱导的小鼠肺纤维化改变显著,Ashcroft评分较正常对照组显著增高(P<0.01),表明小鼠肺纤维化模型构建成功;MMF高剂量干预组肺组织损伤减轻,炎细胞浸润及胶原沉积减少,Ashcroft评分显著降低(P=0.000)。MMF高剂量干预组与BLM模型组比较,TGF-β1基因mRNA的转录水平及蛋白的表达水平均明显减低(P<0.05);而MMF低、中剂量干预组与BLM模型组之间、MMF对照组与正常对照组之间,差异均无统计学意义(P>0.05)。结论高剂量的MMF(100 mg/kg)可抑制BLM诱导的肺纤维化小鼠肺组织中TGF-β1基因mRNA的转录水平及蛋白表达水平,有望成为治疗肺纤维化的理想药物。     

Protective Role of Andrographolide in Bleomycin-Induced Pulmonary Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is a chronic devastating disease with poor prognosis. Multiple pathological processes, including inflammation, epithelial mesenchymal transition (EMT), apoptosis, and oxidative stress, are involved in the pathogenesis of IPF. Recent findings suggested that nuclear factor-κB (NF-κB) is constitutively activated in IPF and acts as a central regulator in the pathogenesis of IPF. The aim of our study was to reveal the value of andrographolide on bleomycin-induced inflammation and fibrosis in mice. The indicated dosages of andrographolide were administered in mice with bleomycin-induced pulmonary fibrosis. On day 21, cell counts of total cells, macrophages, neutrophils and lymphocytes, alone with TNF-α in bronchoalveolar lavage fluid (BALF) were measured. HE staining and Masson’s trichrome (MT) staining were used to observe the histological alterations of lungs. The Ashcroft score and hydroxyproline content of lungs were also measured. TGF-β1 and α-SMA mRNA and protein were analyzed. Activation of NF-κB was determined by western blotting and electrophoretic mobility shift assay (EMSA). On day 21 after bleomycin stimulation, andrographolide dose-dependently inhibited the inflammatory cells and TNF-α in BALF. Meanwhile, our data demonstrated that the Ashcroft score and hydroxyproline content of the bleomycin-stimulated lung were reduced by andrographolide administration. Furthermore, andrographloide suppressed TGF-β1 and α-SMA mRNA and protein expression in bleomycin-induced pulmonary fibrosis. Meanwhile, andrographolide significantly dose-dependently inhibited the ratio of phospho-NF-κB p65/total NF-κB p65 and NF-κB p65 DNA binding activities. Our findings indicate that andrographolide compromised bleomycin-induced pulmonary inflammation and fibrosis possibly through inactivation of NF-κB. Andrographolide holds promise as a novel drug to treat the devastating disease of pulmonary fibrosis.     

Corilagin Attenuates Aerosol Bleomycin-Induced Experimental Lung Injury

Idiopathic pulmonary fibrosis (IPF) is a progressing lethal disease with few clinically effective therapies. Corilagin is a tannin derivative which shows anti-inflammatory and antifibrotics properties and is potentiated in treating IPF. Here, we investigated the effect of corilagin on lung injury following bleomycin exposure in an animal model of pulmonary fibrosis. Corilagin abrogated bleomycin-induced lung fibrosis as assessed by H&E; Masson’s trichrome staining and lung hydroxyproline content in lung tissue. Corilagin reduced the number of apoptotic lung cells and prevented lung epithelial cells from membrane breakdown, effluence of lamellar bodies and thickening of the respiratory membrane. Bleomycin exposure induced expression of MDA, IKKα, phosphorylated IKKα (p-IKKα), NF-κB P65, TNF-α and IL-1β, and reduced I-κB expression in mice lung tissue or in BALF. These changes were reversed by high-dose corilagin (100 mg/kg i.p) more dramatically than by low dose (10 mg/kg i.p). Last, corilagin inhibits TGF-β1 production and α-SMA expression in lung tissue samples. Taken together, these findings confirmed that corilagin attenuates bleomycin-induced epithelial injury and fibrosis via inactivation of oxidative stress, proinflammatory cytokine release and NF-κB and TGF-β1 signaling. Corilagin may serve as a promising therapeutic agent for pulmonary fibrosis.     

Anti-Inflammatory,Antioxidant, and Antifibrotic Effects of Gingival-Derived MSCs on Bleomycin-Induced Pulmonary Fibrosis in Mice

Background: Mesenchymal stem cell (MSC) intervention has been associated with lung protection. We attempted to determine whether mouse gingival-derived mesenchymal stem cells (GMSCs) could protect against bleomycin-induced pulmonary fibrosis. Methods: Mice were divided into three groups: control (Con), bleomycin (Bl), and bleomycin + MSCs (Bl + MSCs). Mice were treated with 5 mg/kg bleomycin via transtracheal instillation to induce pulmonary fibrosis. We assessed the following parameters: histopathological severity of injury in the lung, liver, kidney, and aortic tissues; the degree of pulmonary fibrosis; pulmonary inflammation; pulmonary oedema; profibrotic factor levels in bronchoalveolar lavage fluid (BALF) and lung tissue; oxidative stress-related indicators and apoptotic index in lung tissue; and gene expression levels of IL-1β, IL-8, TNF-α, lysophosphatidic acid (LPA), lysophosphatidic acid receptor 1 (LPA1), TGF-β, matrix metalloproteinase 9 (MMP-9), neutrophil elastase (NE), MPO, and IL-10 in lung tissue. Results: GMSC intervention attenuated bleomycin-induced pulmonary fibrosis, pulmonary inflammation, pulmonary oedema, and apoptosis. Bleomycin instillation notably increased expression levels of the IL-1β, IL-8, TNF-α, LPA, LPA1, TGF-β, MMP-9, NE, and MPO genes and attenuated expression levels of the IL-10 gene in lung tissue, and these effects were reversed by GMSC intervention. Bleomycin instillation notably upregulated MDA and MPO levels and downregulated GSH and SOD levels in lung tissue, and these effects were reversed by GMSC intervention. GMSC intervention prevented upregulation of neutrophil content in the lung, liver, and kidney tissues and the apoptotic index in lung tissue. Conclusions: GMSC intervention exhibits anti-inflammatory and antioxidant capacities. Deleterious accumulation of neutrophils, which is reduced by GMSC intervention, is a key component of bleomycin-induced pulmonary fibrosis. GMSC intervention impairs bleomycin-induced NE, MMP-9, LPA, APL1, and TGF-β release.     

Atorvastatin Attenuates Bleomycin-Induced Pulmonary Fibrosis via Suppressing iNOS Expression and the CTGF (CCN2)/ERK Signaling Pathway

Pulmonary fibrosis is a progressive and fatal lung disorder with high mortality rate. To date, despite the fact that extensive research trials are ongoing, pulmonary fibrosis continues to have a poor response to available medical therapy. Statins, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, known for its broad pharmacological activities, remains a remedy against multiple diseases. The present study investigated the antifibrotic potential of atorvastatin against bleomycin-induced lung fibrosis and to further explore the possible underlying mechanisms. Our results showed that atorvastatin administration significantly ameliorated the bleomycin mediated histological alterations and blocked collagen deposition with parallel reduction in the hydroxyproline level. Atorvastatin reduced malondialdehyde (MDA) level and lung indices. Atorvastatin also markedly decreased the expression of inducible nitric oxide synthase (iNOS) in lung tissues and, thus, prevented nitric oxide (NO) release in response to bleomycin challenge. Furthermore, atorvastatin exhibited target down-regulation of connective tissue growth factor (CTGF (CCN2)) and phosphorylation extracellular regulated protein kinases (p-ERK) expression. Taken together, atorvastatin significantly ameliorated bleomycin-induced pulmonary fibrosis in rats, via the inhibition of iNOS expression and the CTGF (CCN2)/ERK signaling pathway. The present study provides evidence that atorvastatin may be a potential therapeutic reagent for the treatment of lung fibrosis.     

Fenbendazole Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice via Suppression of Fibroblast-to-Myofibroblast Differentiation

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fatal interstitial lung disease with unknown etiology. Despite substantial progress in understanding the pathogenesis of pulmonary fibrosis and drug development, there is still no cure for this devastating disease. Fenbendazole (FBZ) is a benzimidazole compound that is widely used as an anthelmintic agent and recent studies have expanded the scope of its pharmacological effects and application prospect. This study demonstrated that FBZ treatment blunted bleomycin-induced lung fibrosis in mice. In vitro studies showed that FBZ inhibited the proliferation and migration of human embryo lung fibroblasts. Further studies showed that FBZ significantly inhibited glucose consumption, moderated glycolytic metabolism in fibroblasts, thus activated adenosine monophosphate-activated protein kinase (AMPK), and reduced the activation of the mammalian target of rapamycin (mTOR) pathway, thereby inhibiting transforming growth factor-β (TGF-β1)-induced fibroblast-to-myofibroblast differentiation and collagen synthesis. In summary, our data suggested that FBZ has potential as a novel treatment for pulmonary fibrosis.     

Cellular and Molecular Mechanism of Pulmonary Fibrosis Post-COVID-19: Focus on Galectin-1, -3, -8, -9

Pulmonary fibrosis is a consequence of the pathological accumulation of extracellular matrix (ECM), which finally leads to lung scarring. Although the pulmonary fibrogenesis is almost known, the last two years of the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its post effects added new particularities which need to be explored. Many questions remain about how pulmonary fibrotic changes occur within the lungs of COVID-19 patients, and whether the changes will persist long term or are capable of resolving. This review brings together existing knowledge on both COVID-19 and pulmonary fibrosis, starting with the main key players in promoting pulmonary fibrosis, such as alveolar and endothelial cells, fibroblasts, lipofibroblasts, and macrophages. Further, we provide an overview of the main molecular mechanisms driving the fibrotic process in connection with Galactin-1, -3, -8, and -9, together with the currently approved and newly proposed clinical therapeutic solutions given for the treatment of fibrosis, based on their inhibition. The work underlines the particular pathways and processes that may be implicated in pulmonary fibrosis pathogenesis post-SARS-CoV-2 viral infection. The recent data suggest that galectin-1, -3, -8, and -9 could become valuable biomarkers for the diagnosis and prognosis of lung fibrosis post-COVID-19 and promising molecular targets for the development of new and original therapeutic tools to treat the disease.     

Role of rho kinase in microvascular damage following cerebral ischemia reperfusion in rats

Rho kinase (ROCK) is a well-known downstream effector of Rho and plays an important role in various physiopathological processes. In this study, we aim to investigate the correlation between ROCK and microvascular damage in rat brain subjected to middle cerebral artery occlusion (MCAO) and reperfusion, and to elucidate the mechanisms underlying the microvascular damage. ROCK and matrix metalloproteinase 9 (MMP9) mRNA levels were determined by real time quantitative PCR, Laminin was detected by immunofluorescence and Blood Brain Barrier (BBB) permeability was examined by Evans Blue (EB) in rat MCAO models. We observed similar patterns of changes in ROCK expression, brain EB content, and Laminin expression at different time points after brain ischemia. Statistical analysis further confirmed a significant linear correlation of ROCK expression with the onset of microvascular damage in brain. Furthermore, the ROCK inhibitor fasudil decreased brain EB content but increased Laminin expression. These results provide strong evidence that ROCK mediates microvascular damage. In addition, we found that fasudil could significantly inhibit MMP9 expression induced by ischemia. Thus, our findings suggest that ROCK promotes microvascular damage by upregulating MMP9 and reveal ROCK as a promising therapeutic target for stroke.     

Rho/ROCK Pathway and Noncoding RNAs: Implications in Ischemic Stroke and Spinal Cord Injury

Ischemic strokes (IS) and spinal cord injuries (SCI) are major causes of disability. RhoA is a small GTPase protein that activates a downstream effector, ROCK. The up-regulation of the RhoA/ROCK pathway contributes to neuronal apoptosis, neuroinflammation, blood-brain barrier dysfunction, astrogliosis, and axon growth inhibition in IS and SCI. Noncoding RNAs (ncRNAs), such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), were previously considered to be non-functional. However, they have attracted much attention because they play an essential role in regulating gene expression in physiological and pathological conditions. There is growing evidence that ROCK inhibitors, such as fasudil and VX-210, can reduce injury in IS and SCI in animal models and clinical trials. Recently, it has been reported that miRNAs are decreased in IS and SCI, while lncRNAs are increased. Inhibiting the Rho/ROCK pathway with miRNAs alleviates apoptosis, neuroinflammation, oxidative stress, and axon growth inhibition in IS and SCI. Further studies are required to explore the significance of ncRNAs in IS and SCI and to establish new strategies for preventing and treating these devastating diseases.     

Loss of SP-A in the Lung Exacerbates Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating and common chronic lung disease that is pathologically characterized by the destruction of lung architecture and the accumulation of extracellular matrix in the lung. Previous studies have shown an association between lung surfactant protein (SP) and the pathogenesis of IPF, as demonstrated by mutations and the altered expression of SP in patients with IPF. However, the role of SP in the development of lung fibrosis is poorly understood. In this study, the role of surfactant protein A (SP-A) was explored in experimental lung fibrosis induced with a low or high dose of bleomycin (BLM) and CRISPR/Cas9-mediated genetic deletion of SP-A. Our results showed that lung SP-A deficiency in mice promoted the development of fibrotic damage and exacerbated inflammatory responses to the BLM challenge. In vitro experiments with murine lung epithelial LA-4 cells demonstrated that in response to transforming growth factor-β1 (TGF-β1), LA-4 cells had a decreased protein expression of SP-A. Furthermore, exogenous SP administration to LA-4 cells inhibited the TGF-β1-induced upregulation of fibrotic markers. Overall, these findings suggest a novel antifibrotic mechanism of SP-A in the development of lung fibrosis, which indicates the therapeutic potential of the lung SP-A in preventing the development of IPF.     

Nerve Growth Factor: A Potential Therapeutic Target for Lung Diseases

The lungs play a very important role in the human respiratory system. However, many factors can destroy the structure of the lung, causing several lung diseases and, often, serious damage to people’s health. Nerve growth factor (NGF) is a polypeptide which is widely expressed in lung tissues. Under different microenvironments, NGF participates in the occurrence and development of lung diseases by changing protein expression levels and mediating cell function. In this review, we summarize the functions of NGF as well as some potential underlying mechanisms in pulmonary fibrosis (PF), coronavirus disease 2019 (COVID-19), pulmonary hypertension (PH), asthma, chronic obstructive pulmonary disease (COPD), and lung cancer. Furthermore, we highlight that anti-NGF may be used in future therapeutic strategies.     

Pulmonary Fibrosis as a Result of Acute Lung Inflammation: Molecular Mechanisms,Relevant In Vivo Models,Prognostic and Therapeutic Approaches

Pulmonary fibrosis is a chronic progressive lung disease that steadily leads to lung architecture disruption and respiratory failure. The development of pulmonary fibrosis is mostly the result of previous acute lung inflammation, caused by a wide variety of etiological factors, not resolved over time and causing the deposition of fibrotic tissue in the lungs. Despite a long history of study and good coverage of the problem in the scientific literature, the effective therapeutic approaches for pulmonary fibrosis treatment are currently lacking. Thus, the study of the molecular mechanisms underlying the transition from acute lung inflammation to pulmonary fibrosis, and the search for new molecular markers and promising therapeutic targets to prevent pulmonary fibrosis development, remain highly relevant tasks. This review focuses on the etiology, pathogenesis, morphological characteristics and outcomes of acute lung inflammation as a precursor of pulmonary fibrosis; the pathomorphological changes in the lungs during fibrosis development; the known molecular mechanisms and key players of the signaling pathways mediating acute lung inflammation and pulmonary fibrosis, as well as the characteristics of the most common in vivo models of these processes. Moreover, the prognostic markers of acute lung injury severity and pulmonary fibrosis development as well as approved and potential therapeutic approaches suppressing the transition from acute lung inflammation to fibrosis are discussed.     

The Promising Therapeutic Approaches for Radiation-Induced Pulmonary Fibrosis: Targeting Radiation-Induced Mesenchymal Transition of Alveolar Type II Epithelial Cells

Radiation-induced pulmonary fibrosis (RIPF) is a common consequence of radiation for thoracic tumors, and is accompanied by gradual and irreversible organ failure. This severely reduces the survival rate of cancer patients, due to the serious side effects and lack of clinically effective drugs and methods. Radiation-induced pulmonary fibrosis is a dynamic process involving many complicated and varied mechanisms, of which alveolar type II epithelial (AT2) cells are one of the primary target cells, and the epithelial–mesenchymal transition (EMT) of AT2 cells is very relevant in the clinical search for effective targets. Therefore, this review summarizes several important signaling pathways that can induce EMT in AT2 cells, and searches for molecular targets with potential effects on RIPF among them, in order to provide effective therapeutic tools for the clinical prevention and treatment of RIPF.     

Role of Human Primary Renal Fibroblast in TGF-β1-Mediated Fibrosis-Mimicking Devices

Renal fibrosis is a progressive chronic kidney disease that ultimately leads to end-stage renal failure. Despite several approaches to combat renal fibrosis, an experimental model to evaluate currently available drugs is not ideal. We developed fibrosis-mimicking models using three-dimensional (3D) co-culture devices designed with three separate layers of tubule interstitium, namely, epithelial, fibroblastic, and endothelial layers. We introduced human renal proximal tubular epithelial cells (HK-2), human umbilical-vein endothelial cells, and patient-derived renal fibroblasts, and evaluated the effects of transforming growth factor-β (TGF-β) and TGF-β inhibitor treatment on this renal fibrosis model. The expression of the fibrosis marker alpha smooth muscle actin upon TGF-β1 treatment was augmented in monolayer-cultured HK-2 cells in a 3D disease model. In the vascular compartment of renal fibrosis models, the density of vessels was increased and decreased in the TGF-β-treated group and TGF-β-inhibitor treatment group, respectively. Multiplex ELISA using supernatants in the TGF-β-stimulating 3D models showed that pro-inflammatory cytokine and growth factor levels including interleukin-1 beta, tumor necrosis factor alpha, basic fibroblast growth factor, and TGF-β1, TGF-β2, and TGF-β3 were increased, which mimicked the fibrotic microenvironments of human kidneys. This study may enable the construction of a human renal fibrosis-mimicking device model beyond traditional culture experiments.     

COVID-19: Immunohistochemical Analysis of TGF-β Signaling Pathways in Pulmonary Fibrosis

Acute respiratory distress syndrome (ARDS) followed by repair with lung remodeling is observed in COVID-19. These findings can lead to pulmonary terminal fibrosis, a form of irreversible sequelae. There is evidence that TGF-β is intimately involved in the fibrogenic process. When activated, TGF-β promotes the differentiation of fibroblasts into myofibroblasts and regulates the remodeling of the extracellular matrix (ECM). In this sense, the present study evaluated the histopathological features and immunohistochemical biomarkers (ACE-2, AKT-1, Caveolin-1, CD44v6, IL-4, MMP-9, α-SMA, Sphingosine-1, and TGF-β1 tissue expression) involved in the TGF-β1 signaling pathways and pulmonary fibrosis. The study consisted of 24 paraffin lung samples from patients who died of COVID-19 (COVID-19 group), compared to 10 lung samples from patients who died of H1N1pdm09 (H1N1 group) and 11 lung samples from patients who died of different causes, with no lung injury (CONTROL group). In addition to the presence of alveolar septal fibrosis, diffuse alveolar damage (DAD) was found to be significantly increased in the COVID-19 group, associated with a higher density of Collagen I (mature) and III (immature). There was also a significant increase observed in the immunoexpression of tissue biomarkers ACE-2, AKT-1, CD44v6, IL-4, MMP-9, α-SMA, Sphingosine-1, and TGF-β1 in the COVID-19 group. A significantly lower expression of Caveolin-1 was also found in this group. The results suggest the participation of TGF-β pathways in the development process of pulmonary fibrosis. Thus, it would be plausible to consider therapy with TGF-β inhibitors in those patients recovered from COVID-19 to mitigate a possible development of pulmonary fibrosis and its consequences for post-COVID-19 life quality.     

P300/CBP-Associated Factor Activates Cardiac Fibroblasts by SMAD2 Acetylation

Various heart diseases cause cardiac remodeling, which in turn leads to ineffective contraction. Although it is an adaptive response to injury, cardiac fibrosis contributes to this remodeling, for which the reactivation of quiescent myofibroblasts is a key feature. In the present study, we investigated the role of the p300/CBP-associated factor (PCAF), a histone acetyltransferase, in the activation of cardiac fibroblasts. An intraperitoneal (i.p.) injection of a high dose (160 mg/kg) of isoproterenol (ISP) induced cardiac fibrosis and reduced the amount of the PCAF in cardiac fibroblasts in the mouse heart. However, the PCAF activity was significantly increased in cardiac fibroblasts, but not in cardiomyocytes, obtained from ISP-administered mice. An in vitro study using human cardiac fibroblast cells recapitulated the in vivo results; an treatment with transforming growth factor-β1 (TGF-β1) reduced the PCAF, whereas it activated the PCAF in the fibroblasts. PCAF siRNA attenuated the TGF-β1-induced increase in and translocation of fibrosis marker proteins. PCAF siRNA blocked TGF-β1-mediated gel contraction and cell migration. The PCAF directly interacted with and acetylated mothers against decapentaplegic homolog 2 (SMAD2). PCAF siRNA prevented TGF-β1-induced phosphorylation and the nuclear localization of SMAD2. These results suggest that the increase in PCAF activity during cardiac fibrosis may participate in SMAD2 acetylation and thereby in its activation.