Formation Of Pulmonary Fibrosis Research Articles

Prolonged course of novel coronavirus infection COVID-19 with the formation of pulmonary fibrosis in an HIV-infected patient

A description of a clinical case of a severe, protracted course of coronavirus infection (COVID-19) in a patient with advanced stage HIV infection, characterized by prolonged release of the SARS-CoV-2 virus and the formation of pulmonary fibrosis against the background of an active viral infection, is presented. COVID-19 infection occurred in an immunosuppressed patient without ART. During the period of inpatient treatment, a comprehensive examination was carried out to exclude HIV-associated lung lesions; no evidence of viral, fungal, or bacterial pathology was obtained; mycobacterial infections were also excluded. Despite two courses of antiviral therapy, the use of anti-Covid plasma and complex pathogenetic therapy, persistence of SARS-CoV-2 replication was observed for eight months, with a steady progression of the disease, ending in death.

POST-COVID-19 LUNG PATHOGENESIS AND PATHOMORPHOLOGY

The article describes the main pathogenetic and pathomorphological aspects of pulmonary fibrosis onset and development in patients with COVID-19 in anamnesis.
 The authors analyzed open access articles in Russian and English from eLibrary and Pubmed archives.
 The key aspect of the pulmonary fibrosis pathogenesis is fibroblast and myofibroblast activation. In response to the lung parenchyma damage, it leads to fibroblast and myofibroblast proliferation and differentiation and triggers a cascade of cytokine reactions. T-helper cells are responsible for the regulation of the inflammatory-reparative process in the lungs. T-helper cells directly or indirectly trigger the remodeling of the pulmonary parenchyma in favor of the fibrous component.
 
 Literature shows that the role of cytokines is assessed differently, and currently there is no consensus on their influence on pulmonary fibrosis formation. However, studies showing the possibility to prevent and treat fibrosis with anti-cytokine drugs place the development of a cytokine storm at the forefront.
 Growth factors, especially TGF, FGF, PDGF, are important not only in understanding pathogenesis, but also in finding new, promising therapeutic modalities.
 Due to external factors, many authors refrain from quantitative assessments of long-term consequences. Data on the persistence and regression of post-Covid pulmonary fibrosis are also contradictory.
 Despite much information on issues related to COVID-19 pathogenesis and pulmonary fibrosis development, many molecular mechanisms remain hidden from researchers. Thus, there are new prospects in diagnosis, prevention and treatment of the disease.

Progress in Understanding the Role and Therapeutic Targets of Polarized Subtypes of Macrophages in Pulmonary Fibrosis.

Pulmonary fibrosis represents the advanced phase of diverse pulmonary ailments, and at present, a definitive cure for these ailments is lacking. Furthermore, underlying mechanisms causative of these ailments remain elusive. Macrophages are immune cells that resist external stimuli in the early stages after birth. These cells can polarize into the classically (M1) and alternatively (M2) activated macrophages. When stimulated owing to the presence of toxic factors, M1 macrophages produce several pro-inflammatory factors, which mediate the inflammatory injury response of the alveolar tissue. The secretion of diverse growth factors by M2 macrophages contributes to the pathogenesis of aberrant alveolar structural fibrosis and remodeling. The abnormal activity of M2 macrophages is considered a critical factor in the formation of pulmonary fibrosis. In this mini-review, to highlight the clinical implications of research studies, we summarize the role and therapeutic targets of polarized subtypes of macrophages in pulmonary fibrosis and the role of targeting macrophages for the treatment of pulmonary fibrosis.

Indicators of lung function in children with chronic nonspecific lung diseases with pulmonary fibrosis

Introduction. The pathogenesis of most chronic nonspecific lung diseases (CNSLD) in children is based on a long-term inflammatory process, often leading to the formation of pulmonary fibrosis in the structurally altered tissue, which requires dynamic monitoring of patients, including the study of lung function.Aim. To assess the indicators of the lung function in children with chronic bronchopulmonary pathology accompanied by pulmonary fibrosis.Materials and methods. 84 children with CNSLD were examined. The main group consisted of 45 children with CNSLD with pulmonary fibrosis (bronchopulmonary dysplasia, congenital malformations of the lungs, chronic bronchitis, and post pneumonia fibrosis). The comparison group consisted of 39 children with CNSLD without pulmonary fibrosis. The average age of children in the study groups was 9,3±0,48 years. The patients underwent multispiral computed tomography of the lungs with a virtual bronchoscopy program and intravenous bolus contrast enhancement. Evaluation of lung function was carried out by spirometry.Results. In patients in remission, the average values of the ventilation capacity of the lungs in both groups were within the predicted values. However, in patients with fibrotic changes, pulmonary dysfunction was detected 2 times more often than in children with CNSLD without pulmonary fibrosis (35.5% and 15.4%, respectively, p<0.05). Restrictive ventilation disorders were diagnosed only in the group of patients with pulmonary fibrosis. In children with pulmonary fibrosis, the risk of reduced ventilation capacity is significantly higher than in children with CNSLD without pulmonary fibrosis (OR=3.04, 95% CI 1.049–8.78).Conclusion. The data obtained can serve as a prerequisite for further development of predicting the nature of the course of the disease, identifying “risk groups” for the development of fibrotic changes for a personalized approach to the treatment and follow-up of patients with CNSLD.

Disturbance of serum lipid metabolites and potential biomarkers in the Bleomycin model of pulmonary fibrosis in young mice

BackgroundAltered metabolic pathways have recently been considered as potential drivers of idiopathic pulmonary fibrosis (IPF) for the study of drug therapeutic targets. However, our understanding of the metabolite profile during IPF formation is lacking.MethodsTo comprehensively characterize the metabolic disorders of IPF, a mouse IPF model was constructed by intratracheal injection of bleomycin into C57BL/6J male mice, and lung tissues from IPF mice at 7 days, 14 days, and controls were analyzed by pathology, immunohistochemistry, and Western Blots. Meanwhile, serum metabolite detections were conducted in IPF mice using LC–ESI–MS/MS, KEGG metabolic pathway analysis was applied to the differential metabolites, and biomarkers were screened using machine learning algorithms.ResultsWe analyzed the levels of 1465 metabolites and found that more than one-third of the metabolites were altered during IPF formation. There were 504 and 565 metabolites that differed between M7 and M14 and controls, respectively, while 201 differential metabolites were found between M7 and M14. In IPF mouse sera, about 80% of differential metabolite expression was downregulated. Lipids accounted for more than 80% of the differential metabolite species with down-regulated expression. The KEGG pathway enrichment analysis of differential metabolites was mainly enriched to pathways such as the metabolism of glycerolipids and glycerophospholipids. Eight metabolites were screened by a machine learning random forest model, and receiver operating characteristic curves (ROC) assessed them as ideal diagnostic tools.ConclusionsIn conclusion, we have identified disturbances in serum lipid metabolism associated with the formation of pulmonary fibrosis, contributing to the understanding of the pathogenesis of pulmonary fibrosis.

Chronic fibrosing interstitial lung diseases according to the register of Irkutsk (Russia)

The Interstitial lung diseases (ILD) are a wide heterogeneous group of disorders, different in many parameters, however having similar pathophysiological mechanisms. Idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia (NSIP), chronic hypersensitivity pneumonitis (CHP), ILD associated with autoimmune disorders (predominantly systemic sclerosis (SSc ILD) and rheumatoid arthritis (RA ILD), and other diseases may be accompanied by the formation of pulmonary fibrosis. Currently there are no generally accepted criteria for the fibrosing and progressive fibrosing ILD, and the information on the frequency of these conditions is limited.The study aims to research the structure of fibrosing ILD and the frequency of the progressive fibrosing phenotype (PFP) according to the register of patients with ILD in Irkutsk, Russia.Methods. At the first stage information letters were sent to all medical institutions of Irkutsk with recommendations on all referral patients with ILD for further examination. At the second stage, each ILD case was verified collegially (n = 270). All patients’ data were analysed in terms of clinical, functional, radiological and histological aspects. A register of ILD patients was formed based on this analysis. The follow-up period was 1 to 5 years.Results. According to the results of a comprehensive assessment, HRCT signs of pulmonary fibrosis were detected by 104 patients (38.5%). fibrosing course was observed in 100% IPF and SSc ILD, in 90.9% of CHP, 71.4% of NSIP and 60% of RA ILD. 61 patients (22.6% of all ILD cases, 58.6% of fibrosing ILD) was diagnosed with PFP. The 5-year mortality for PFP ILD was 55.5%.Conclusion. In the case of a PFP development, the evolution of ILD loses its nosological specificity, acquiring similarity with IPF. Due to the high mortality rate, it is necessary to actively identify the progressive fibrosing course of a wide range of ILD and to prescribe an antifibrotic therapy in a timely manner.

Coronavirus disease 2019-associated pulmonary fibrosis: clinical findings, pathogenesis, and potential treatment.

Coronavirus disease 2019-associated pulmonary fibrosis: clinical findings, pathogenesis, and potential treatment.

The Ameliorative Effects of Arctiin and Arctigenin on the Oxidative Injury of Lung Induced by Silica via TLR-4/NLRP3/TGF-β Signaling Pathway.

Silicosis remains one of the most serious diseases worldwide, with no effective drug for its treatment. Our research results have indicated that arctiin and arctigenin could increase the mitochondrial membrane potential, which in turn reduces the production of reactive oxygen species (ROS), blocks the polarization of macrophages, and inhibits the differentiation of myofibroblasts to reduce oxidative stress, inflammation, and fibrosis. Further, our study revealed that arctiin and arctigenin suppressed the activation of NLRP3 inflammasome through the TLR-4/Myd88/NF-κB pathway and the silica-induced secretion of TNF-α, IL-1β, TGF-β, and α-SMA. Besides, the silica-induced increase in the levels of serum ceruloplasmin and HYP was also inhibited. Results of metabolomics indicated that arctiin and arctigenin could regulate the abnormal metabolic pathways associated with the development of silicosis, which involve pantothenate and CoA biosynthesis, cysteine and methionine metabolism, linoleic acid metabolism, and arginine and proline metabolism successively. Furthermore, the analysis of metabolomics, together with network topological analysis in different phases of silicosis, revealed that urine myristic acid, serum 4-hydroxyproline, and L-arginine could be regarded as diagnosis biomarkers in the early phase and formation of pulmonary fibrosis in the latter phases of silicosis. Arctiin and arctigenin could downregulate the increased levels of myristic acid in the early phase and serum 4-hydroxyproline in the latter phase of silicosis. Interestingly, the integration of TLR-4/NLRP3/TGF-β signaling and metabolomics verified the importance of macrophage polarization in the silicosis fibrosis process. To the best of our knowledge, this is the first study reporting that arctiin and arctigenin both can ameliorate silicosis effectively, and the former is a little stronger than its aglycone arctigenin because of its high oral bioavailability, low toxicity, and multimolecular active metabolites as determined by AdmetSAR and molecular docking analysis.

The Improvement Effect of Sodium Ferulate on the Formation of Pulmonary Fibrosis in Silicosis Mice Through the Neutrophil Alkaline Phosphatase 3 (NALP3)/Transforming Growth Factor-β1 (TGF-β1)/α-Smooth Muscle Actin (α-SMA) Pathway.

BackgroundPneumoconiosis is a chronic progressive fibrotic interstitial pneumonia for which the pathogenesis and treatment remain unclear. Previous studies showed that sodium ferulate (SF) may have a therapeutic effect, and this study explored the mechanism underlying SF-related improvement.Material/MethodsIn this study, a silicosis mouse model and primary cultured mouse lung fibroblasts were established. Hematoxylin-eosin staining, western blot analysis, quantitative real-time polymerase chain reaction, and Masson staining were used to observe the lung injury, expression of vimentin, and the degree of pulmonary fibrosis. The extracted lung fibroblasts were identified by immunofluorescence. The expression of fibrosis-related genes encoding transforming growth factor-β1 (TGF-β1), neutrophil alkaline phosphatase 3 (NALP3), collagen-1, α-smooth muscle actin (α-SMA), and phosphorylated p38 (p-p38) and p38 proteins were detected by western blot. The effects of SF and the TGF-β pathway agonist SRI-011381 on cell proliferation and the expression of fibrosis-related protein in mouse lung fibroblasts were measured by Cell Counting Kit-8, immunofluorescence, and western blot as needed.ResultsSF reduced the lung lesions in silicosis mice and inhibited the expression of vimentin and fibrosis-related genes, while having no effect on body weight. Vimentin expression was positive in the extracted cells. In vitro experiments showed that SF inhibited the proliferation of lung fibroblasts and the expression of fibrosis-related proteins. In addition, SF partly reversed the opposite regulatory effect of SRI-011381 on lung fibroblasts.ConclusionsSF inhibited lung injury and fibrosis in silicosis mice through the NALP3/TGF-β1/α-SMA pathway.

INVESTIGATION OF EXTERNAL RESPIRATORY FUNCTION AND HEMOSTASIS SYSTEM’S STATUS IN PATIENTS WITH CHRONIC OBSTRUCTIVE LUNG DISEASE WITH CONCOMITANT CHRONIC PANCREATITIS

INTRODUCTION: It can be assumed that the comorbidity course of сhronic obstructive pulmonary disease (COPD) and chronic pancreatitis (CP) can enhance the clinical symptoms of both diseases and lead to frequent relapses of the pathological process due to changes in the proteolysis intensity of high and low molecular weight plasma proteins and the state of the hemocoagulation.
 OBJECTIVE: To establish the features of hemocoagulation and proteolytic hemostasis at COPD with concomitant CP.
 METHODS: 120 patients with COPD and CP were examined. The function of external respiration, total coagulation potential of blood plasma, the state of enzymatic and non-enzymatic fibrinolysis, total fibrinolytic activity were investigated.
 RESULTS: CP contributes to the development of broncho-obstructive syndrome, and the maximum indicators of reduction of FEV1 relative to the proper values are observed in patients with a comorbid course of COPD and CP. Reducing the intensity of collagenolysis in patients of groups 1-2 contributed to the development of diffuse pulmonary fibrosis in response to chronic inflammation. The imbalanced increase in the intensity of proteolysis due to reduced expression of its inhibitors in COPD patients with CP led to progressive destruction of the cell membranes of alveolocytes, acinar epithelium of pancreas and epithelium of the bronchial mucosa, acceleration of their apoptosis and development of desquamation, atrophic changes, metaplasia, and the like. The above factors are active as inducers of inflammation, and the formation of pulmonary fibrosis and fibrosis of the pancreas.
 CONCLUSIONS: In COPD patients with accompanying CP are an increase in the lysis rate of low and high molecular weight proteins and a decrease in blood collagenolytic activity on the background of a significant imbalance in the activity of plasma proteinase inhibitors. Defined suppression of the activity of the anti-coagulation system factors and enzymatic, Hageman-factor-dependent fibrinolysis indicates the formation of hypercoagulation syndrome in these patients.

An animal research and a chemical composition analysis of a Chinese prescription for pulmonary fibrosis: Yangfei Huoxue Decoction

Ethnopharmacological evidencePulmonary fibrosis (PF) is a progressive disease characterized by the aberrant accumulation of fibrotic tissue in the lungs parenchyma, associated with significant morbidity. Few effective drugs have been developed to reverse PF or even halt the disease progression. Yangfei Huoxue Decoction (YHD), a Traditional Chinese Medicine, which consisted of Astragalus membranacus(AM), Glehnia littoralis(GL), Schisandra chinensis(SC), Salvia miltiorrhiza Bunge(SB), Reynoutria japonica(RJ), Ligusticum chuanxiong(LX), and Euonymus alatus(EA) , has been used in China for the treatment of PF for many years with remarkable efficacy.According to the clinic observation of the results, we conducted experiments on animals, the process of BLM-induced pulmonary fibrosis in rats was interfered by YHD, through the detection of pulmonary fibrosis rats’ blood cells and plasma, we selected the related molecules that may exert proinflammatory(IL-1β), promote angiogenesis(vascular endothelial growth factor ,VEGF).For further explicitly research, we should know what the chemical composition the prescription (YHD) contains and what the related bioactive components have.In accordance with in-house library and evaluating the characteristic MS fragmentation patterns, the schisandra chinensis methanol, lignin, flavonol, polyphenol, tanshinone, salvianolic acid, anthraquinone, ligustrazine, etc. had a retardant and inhibitory effect on the development and formation of pulmonary fibrosis. These results will aid in the quality control of YHD, as well as provide fundamental data for further pharmaco-mechanisms studies. Aim of the studyTo discover the pulmonary immune related bioactive components of YHD. Materials and methodsAnimal Experiment:144 SD rats, based on the principles of randomization divided into eight groups, Control group, bleomycin(BLM) group, BLM + dexamethasone(BLM + DXM) group, BLM + Yangfei(YF) group, BLM + Huoxue(HX) group, BLM + high-doseYHD(YHD-H) group, BLM + medium-doseYHD(YHD-M) group, and BLM + low-doseYHD(YHD-L) group, each group of 18 rats. After endotracheal administration of Bleomycin by tracheotomy, rats were sacrificed on day 7, day 14 and day 28, blood and plasma were taken at the same time. Respectively, the VEGF, an immune molecule associated with angiogenesis, and IL-1β in plasma were detected by ELISA at three time periods. Component testing: 100 g YHD were constituted of SB 15 g, LX 12 g, EA 10 g, RJ 15 g, AM 20 g, GL 20 g and SC 8 g. All herbs were obtained from Beijing Tong Ren Tang (Group) Co ltd. The voucher specimens were identified by Prof. Jiening Gong (Nanjing University of Chinese Medicine). YHD were extracted by sonication with 1 L ethanol/water (70:30, v/v) for two cycle (1 h per cycle) at room temperature. The combined extracts were filtered, condensed, and reconstituted with 50 mL methanol before analysis. Standard Cianidanol, Ferulic Acid, Polydatin, Calycosin 7-O-glucoside, Tanshinone IIA, Salvianolic acid B, Schizandrol A, and Isoimperatorin were prepared in methanol. After centrifuging at 20,000 rpm for 10 min, 4 μL supernatant was injected into the Ultra-Performance Liquid Chromatography coupled with Quadrupole Time-of-Flight tandem mass spectrometry (UPLC/QTOF-MSE) combined with UNIFI informatics platform for analysis. ConclusionThe experiment results revealed that the vascularized VEGF, inflammatory factor expression of IL-1β was restrained by YHD.The UPLC/QTOF-MSE method, an automatic database screening platform and the characteristic MS fragmentation patterns have efficiently facilitated the post data process, so we test for the identification of major components in YHD by this technology, more than seven or more active ingredients, the results showed that YHD contained a total of 55 components, including 11 lignans, 12 flavonoids, 7 tanshinones, 9 organic acid, 5 polyphenols, 4 anthraquinones, 5 senkyunolides and 2 others. Based on this, we can ensure the discovery and analysis of biologically active compounds in YHD, as well as provide a reference for the quality evaluation. We expect the method presented here could be applied to other multi-component TCM formula. In addition, we can conduct more in-depth research, such as mechanism research, molecular detection, gene target and so on.

Effect of caffeine on hyperoxic lung injury of premature rats and its relationship with p38 motigen-activated protein kinase signal pathway

Objective To study the effect of caffeine on hyperoxic lung injury of premature rats and its relationship with p38 motigen-activated protein kinase(MAPK) signal pathway. Methods Sixty Wistar premature rats were divided into 4 groups(n=15) according to the random number table: air + normal saline group(A+ N group), air + caffeine group(A+ C group), hyperoxia + normal saline group(H+ N group), and hyperoxia + caffeine group(H+ C group). Among them, H+ N group and H+ C group were continually exposed to hyperoxia(oxygen concentration was 60%~70%). For A+ C group and H+ C group, the premature rats were injected with caffeine of 29 mg/(kg·d) into their peritoneal cavities every day after birth.For A+ N group and H+ N group, the premature rats were injected with normal saline of the same volume into their peritoneal cavities.In each group, the lung tissues of 5 premature rats were randomly selected on the third, seventh and fourteenth day respectively.The pathological changes of lung tissue, radiated alveolar count(RAC) and collagen content in lung tissue were observed under a light microscope.The wet/dry ratio(W/D) was measured.Two-step immunohistochemistry was used to detect the distribution of p38MAPK in lung tissue.The content of phosphorylated p38MAPK(p-p38MAPK) protein was detected by western blot. Results Compared with the air groups, the lung tissues of premature rats in high oxygen exposure groups showed different degree of inflammatory changes on the third, seventh, and fourteenth day.The changes were more obvious with the prolonged exposure to hyperoxia.Pulmonary fibrosis was visible on the fourteenth day, which was improved after caffeine intervention.The RAC value of premature rats in hyperoxia exposure groups was significantly lower than that in air-exposure groups(P<0.05), and the W/D ratio and collagen content in lung tissue increased significantly(P<0.05), which were improved after caffeine intervention(P<0.05). The results of two-step immunohistochemistry showed that the number of p-p38MAPK positive cells in the lung tissue of premature rats in hyperoxia exposure groups increased and widely distributed, but decreased after caffeine intervention.The results of western blot showed that the content of p-p38MAPK protein in lung tissue of premature rats in hyperoxia exposure groups was significantly higher than that of air groups(P<0.05), but it decreased after caffeine intervention(P<0.05). Conclusion Hyperoxia can promote the formation of pulmonary fibrosis by activating p38MAPK signal pathway.Caffeine can interdict the expression of p38MAPK to alleviate the fibrosis of lung tissue exposed to hyperoxia and thus protects the lung tissue. Key words: Hyperoxia; Lung injury; Caffeine; p38 motigen-activated protein kinase; Premature rats

Expression of TRB3 promotes epithelial‑mesenchymal transition of MLE‑12 murine alveolar type II epithelial cells through the TGF‑β1/Smad3 signaling pathway.

The aim of the present study was to investigate whether the expression of tribbles pseudokinase 3 (TRB3) is involved in pulmonary interstitial fibrosis and to examine the possible mechanisms. The expression of TRB3 in murine alveolar type II epithelial cells (MLE‑12 cells) following transforming growth factor β1 (TGF‑β1) stimulation was assessed using various techniques, including western blot and reverse transcription‑quantitative polymerase chain reaction assays. TRB3 overexpression and downregulation models were used to evaluate the impact of TRB3 on the TGF‑β1‑induced epithelial‑mesenchymal transition (EMT) of MLE‑12 cells. The downregulation of TRB3 was induced by RNA interference. The expression of TRB3 was significantly increased in MLE‑12 cells following the activation of TGF‑β1 (P<0.05). The overexpression of TRB3 was found to promote activation of the TGF‑β1/Smad3 signaling pathway, EMT, and the upregulated expression of β‑catenin and EMT‑related genes and proteins (P<0.05), whereas the downregulation of TRB3 attenuated the promoting effect on EMT induced by TGF‑β1. In addition, the overexpression of TRB3 inhibited MLE‑12 cell proliferation by stimulating apoptosis, leading to the formation of pulmonary fibrosis (PF). The positive feedback loop demonstrated that TGF‑β1 induced the expression of TRB3, and TRB3, in turn, stimulated EMT and promoted the onset of PF through activation of the TGF‑β1/Smad3 signaling pathway. Therefore, TRB3 may promote the formation of PF through the TGF‑β1/Smad3 signaling pathway.

Downregulation of HMGB1 is required for the protective role of Nrf2 in EMT‐mediated PF

Epithelial-mesenchymal transition (EMT) is considered to be the key event in the formation of pulmonary fibrosis (PF). High-mobility group box 1 (HMGB1) is a novel mediator of EMT. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical transcription factor for protecting against PF. However, it is unknown the relationship between Nrf2 and HMGB1 in EMT-mediated PF. Bleomycin (BLM)-induced PF in Nrf2-knockout (Nrf2-/- ) and wild-type (WT) mice and transforming growth factor β1 (TGF-β1)-induced EMT in rat type II alveolar epithelial cell line (RLE-6TN) and human alveolar epithelial cell line (A549) were established to observe the relationship among Nrf2, HMGB1, and EMT by western blot and immunohistochemistry. BLM-induced EMT was more severe and the expression of HMGB1 was more increased in Nrf2 -/- mice compared with WT mice. In vitro, Nrf2 activation attenuated TGF-β1-induced EMT and ROS production accompanied by the downregulation of HMGB1. In contrast, silencing Nrf2 enhanced TGF-β1-induced EMT and ROS production along with increased the protein expression and the release of HMGB1. Moreover, HMGB1 activation aggravated TGF-β1-induced EMT and HMGB1 deficiency alleviated TGF-β1-induced EMT. Furthermore, HMGB1 silence attenuated the protective effect of Nrf2 on EMT. These findings suggest downregulation of HMGB1, which is required for the protective role of Nrf2 in EMT-mediated PF and provide an important therapeutic target for PF.

ADAM17/EGFR-dependent ERK activation mediates thrombin-induced CTGF expression in human lung fibroblasts

Lung fibroblasts play critical roles in fibrotic procedures and contribute to lung fibrosis. Several studies indicated that thrombin, a disintegrin and metalloproteinase 17 (ADAM17), and connective tissue growth factor (CTGF) participate in the formation of pulmonary fibrosis. In this study, we examined the involvement of the ADAM17/epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase (ERK) pathway in thrombin-stimulated CTGF manifestation in human lung fibroblasts (WI-38). We found that cells treated with thrombin significantly increased ADAM17 expression, ADAM17 and c-Jun phosphorylation in time-dependent manners. Thrombin-stimulated CTGF expression, ERK and c-Jun phosphorylation were inhibited by TAPI-0 (an ADAM17 inhibitor). Moreover, U0126 (an ERK inhibitor) inhibited thrombin-stimulated CTGF expression and c-Jun phosphorylation. Cells transfected with small interfering RNA of the EGFR attenuated thrombin-stimulated ERK phosphorylation, c-Jun phosphorylation, and CTGF expression. Thus, these results suggested that ADAM17/EGFR-dependent ERK activation mediated thrombin-stimulated CTGF expression in human lung fibroblasts.

Examination of the expressions of miR-221 in the human pulmonary fibrosis tissues and in A549 cells treated with TGFβ1

Objective To investigate the expressions of microRNA-221 (miR-221) in the human pulmonary fibrosis tissues and in adenocarcinoma(A549) cells treated with transforming growth factor beta 1 (TGFβ1). Methods Real time-PCR was used to detect the expressions of miR-221 in pathologically diagnosed as pulmonary fibrosis tissue and in A549 cells treated with TGFβ1. Results miR-221 in pulmonary fibrosis tissue was downregulated compared to normal tissues (P<0.05). The morphology of cells was changed from long spindle type to short, the number of cells was increased, and the adjacent cells were not connected closely. TGFβ1, N-cadherin, vimentin, α-smooth muscle actin (α-SMA), collagen Ⅰ, and collagen Ⅲ levels were higher, whereas E-cadherin level was lower in TGFβ1-treated group compared to the control group. miR-221 expression was downregulated in cells after TGFβ1 treatment (P<0.05). Conclusions miR-221 was downregulated in pulmonary fibrosis tissues and in A549 cells, which indicates that miR-221 may play an important role in the formation of pulmonary fibrosis. Key words: MicroRNAs/ME; Pulmonary fibrosis/PA; Transforming growth factor beta1/AE; Lung neoplasms/PA

O29 Nrf2/HO-1 pathway alleviates epithelial-mesenchymal transition by inhibiting HMGB1 expression in pulmonary fibrosis

Pulmonary fibrosis (PF) is a chronic, interstitial lung disease with unknown cause, characterized by the activation of accumulated myofibroblasts and deposition of extracellular matrix (ECM). Epithelial-mesenchymal transition (EMT) is considered to be one of the major hypotheses behind the formation of PF. High mobility group box1 (HMGB1) plays an important biological role in infection, inflammation, and immune responses. Nuclear factor E2-related factor 2 (Nrf2) is an important transcription factor for the regulation of oxidative stress. However, there is no direct evidence regarding the relationship between the Nrf2/HO-1 antioxidant system and HMGB1 in lung fibrosis. In our studies, the PF model was induced with bleomycin in both wild type (WT) mice and Nrf2 −/− mice. Rat type II alveolar epithelial cells (RLE-6TN) and the human alveolar epithelial cell line A549 were treated with the Nrf2 activator, sulforaphane, or transfected with Nrf2 and HMGB1 siRNAs to explore their relationship in TGF-β-induced PF. This research showed that bleomycin-induced fibrosis was more severe in Nrf2 −/− mice compared to WT mice. In the in vitro experiments, sulforaphane-treated cells had significantly attenuated TGF-β-induced epithelial-mesenchymal transition accompanied by downregulated expression of HMGB1. In contrast, silencing Nrf2 by siRNA enhanced TGFβ1-induced EMT with increased expression of HMGB1. However, when HMGB1 was silenced by siRNA, sulforaphane could not reduce the progression of EMT in either RLE-6TN cells or A549 cells. These findings suggest that the inhibitory effect of HMGB1 on EMT in PF is regulated by the Nrf2/HO-1 antioxidant pathway.

Reduced Supply of Monocyte-Derived Macrophages Leads to a Transition from Nodular to Diffuse Lesions and Tissue Cell Activation in Silica-Induced Pulmonary Fibrosis in Mice

Pulmonary fibrosis (PF) is an intractable disorder with a poor prognosis. Lung macrophages have been reported to regulate both progression and remission of bleomycin-induced diffuse PF. However, it remains unclear how macrophages contribute to silica-induced progressive nodular PF and the associated tissue cell responses invivo. We found that lack of monocyte-derived macrophages results in the formation of diffuse PF after silica instillation. We found that the proportion and the number of monocyte-derived macrophages were persistently higher in silica-induced progressive PF compared with bleomycin-induced PF. Surprisingly, in Ccr2(-/-) mice, in which monocyte-derived macrophage infiltration is impaired, silica administration induced diffuse PF with loose nodule formation and greater activation of tissue cells. In the diffuse lesions, the distribution of epithelial cells, distribution of myofibroblasts, and architecture of the basement membrane were disrupted. Consistent with the development of diffuselesions, genes that were differentially expressed in CD45(-) tissue cells from the lung of wild-type and Ccr2(-/-) mice were highly enriched in human diffuse, progressive PF. In gene ontology network analyses, many of these genes were associated with tissue remodeling and included genes not previously associated with PF, such as Mmp14, Thbs2, andFgfr4. Overall, these results indicate that monocyte-derived macrophages prevent transition from nodular to diffuse silica-induced PF, potentially by regulating tissue cell responses.

The interventional effect of Xuebijing injection on expression of mitochondrial fusion protein 2 and the ultrastructure changes in lung tissues in rats with paraquat poisoning

To investigate the mechanism of pulmonary fibrosis induced by paraquat (PQ), and the effect of Xuebijing injection in treatment of PQ poisoning. Seventy-two male Wistar rats were randomly divided into control group, PQ poisoning group, and Xuebijing intervention group, with 24 rats in each group. Pulmonary fibrosis was induced by single gavage at the dosage of 50 mg/kg of PQ, while 1 mL of distilled water was given by gavage in control group. Xuebijing injection at the dosage of 4 mL/kg were given intraperitoneally at 30 minutes after exposure to PQ in Xuebijing group, and it was repeated every 12 hours; same amount of physiological saline was given intraperitoneally in PQ group and control group. The experiment lasted for 14 days. Six rats in each group were sacrificed on 1, 3, 7, 14 days, respectively, after insult, and 30 minutes after the last intervention. The lung tissues were harvested, the changes in pathology in lung tissue and the degree of pulmonary fibrosis were observed with optical microscope with hematoxylin-eosin (HE) staining and Masson stain. The ultrastructure changes in lung tissues were observed with transmission electron microscopic, and the content of hydroxyproline (HYP) in the lung tissue was determined by alkaline hydrolysis. The expression of mitochondrial fusion protein 2 (Mfn2) was determined by Western Blot. (1) HE staining: in PQ group, inflammation was most marked on the 3rd day. On the 7th day, exudates in the alveoli started to be organized, and hypertrophic fibroblasts were seen to secrete slim collagen fibers, and fibrosis could be seen in alveoli. On the 14th day, intensive hyperplasia of fibroblasts could be observed, and the alveolar structure was destroyed and collapsed, with deposition of collagen deposited with formation of pulmonary fibrosis. At the same time, pathologic changes were milder in Xuebijing group than those in PQ group. (2) Masson staining: the degree of inflammation in alveoli and pulmonary fibrosis were less marked in Xuebijing group than those of PQ group on the 14th day. (3) Under the transmission electron microscopy, it was found that the mitochondria of lung tissue cells was relatively less in number on the 14th day in PQ group, and the majority of them underwent degeneration, swelling and damage. Basement membrane became folded, alveoli were collapsed, and fibrosis was obvious. These changes were less serious in Xuebijing group. (4) Content of HYP: contents of HYP in lung tissues on the 3rd day in PQ group and Xuebijing group were significantly higher than those in control group (743.3±50.2 μg/g, 718.1±34.0 μg/g vs. 665.8±6.6 μg/g, both P<0.05), it then increased gradually, but the contents of HYP in Xuebijing group were significantly lower on the 7th day and 14th day than those in PQ group (790.5±23.8 μg/g vs. 876.7±42.0 μg/g, 812.9±72.3 μg/g vs. 931.3±33.0 μg/g, both P<0.05). (5) Expression of Mfn2: the expression of Mfn2 in control group was relatively lower. The expression of Mfn2 in PQ group was increased gradually under stress, but its rate was low. The expression of Mfn2 (A value) in Xuebijing group was significantly higher than that in PQ group on the 1st day (0.731±0.035 vs. 0.618±0.029, P<0.05), and it was elevated steadily, reaching the peak on the 7th day (0.732±0.037 vs. 0.669±0.034, P<0.05), but it was lower than that of PQ group on the 14th day (0.708±0.034 vs. 0.765±0.041, P<0.05). Xuebijing reduces lung inflammatory reaction and pulmonary fibrosis as a result of PQ poisoning. The mechanism is that Xuebijing regulates and increases expression of Mfn2 in lung tissue.

Pulmonary fibrosis induced by H5N1 viral infection in mice

BackgroundInflammatory process results in lung injury that may lead to pulmonary fibrosis (PF). Here, we described PF in mice infected with H5N1 virus.MethodsEight-week-old BALB/c mice were inoculated intranasally with 1 × 101 MID50 of A/Chicken/Hebei/108/2002(H5N1) viruses. Lung injury/fibrosis was evaluated by observation of hydroxyproline concentrations, lung indexes, and histopathology on days 7, 14, and 30 postinoculation.ResultsH5N1-inoculated mice presented two stages of pulmonary disease over a 30-d period after infection. At acute stage, infected-mice showed typical diffuse pneumonia with inflammatory cellular infiltration, alveolar and interstitial edema and hemorrhage on day 7 postinoculation. At restoration stage, most infected-mice developed PF of different severities on day 30 postinoculation, and 18% of the survived mice underwent severe interstitial and intra-alveolar fibrosis with thickened alveolar walls, collapsed alveoli and large fibrotic areas. The dramatically elevated hydroxyproline levels in H5N1-infected mice showed deposition of collagen in lungs, and confirmed fibrosis of lungs. The dry lung-to-body weight ratio was significantly increased in infected group, which might be associated with the formation of PF in H5N1-infected mice.ConclusionOur findings show that H5N1-infected mice develop the typical PF during restoration period, which will contribute to the investigation of fibrogenesis and potential therapeutic intervention in human H5N1 disease.